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  • 1.
    Behra, Phani Rama Krishna
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Das, Sarbashis
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Pettersson, B. M. Fredrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Shirreff, Lisa
    Univ Louisiana, Dept Biol, Lafayette, LA USA.
    DuCote, Tanner
    Univ Louisiana, Dept Biol, Lafayette, LA USA.
    Jacobsson, Karl-Gustav
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Ennis, Don G.
    Univ Louisiana, Dept Biol, Lafayette, LA USA.
    Kirsebom, Leif A
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Extended insight into the Mycobacterium chelonae-abscessus complex through whole genome sequencing of Mycobacterium salmoniphilum outbreak and Mycobacterium salmoniphilum-like strains2019In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 4603Article in journal (Refereed)
    Abstract [en]

    Members of the Mycobacterium chelonae-abscessus complex (MCAC) are close to the mycobacterial ancestor and includes both human, animal and fish pathogens. We present the genomes of 14 members of this complex: the complete genomes of Mycobacterium salmoniphilum and Mycobacterium chelonae type strains, seven M. salmoniphilum isolates, and five M. salmoniphilum-like strains including strains isolated during an outbreak in an animal facility at Uppsala University. Average nucleotide identity (ANI) analysis and core gene phylogeny revealed that the M. salmoniphilum-like strains are variants of the human pathogen Mycobacterium franklinii and phylogenetically close to Mycobacterium abscessus. Our data further suggested that M. salmoniphilum separates into three branches named group I, II and III with the M. salmoniphilum type strain belonging to group II. Among predicted virulence factors, the presence of phospholipase C (plcC), which is a major virulence factor that makes M. abscessus highly cytotoxic to mouse macrophages, and that M. franklinii originally was isolated from infected humans make it plausible that the outbreak in the animal facility was caused by a M. salmoniphilum-like strain. Interestingly, M. salmoniphilum-like was isolated from tap water suggesting that it can be present in the environment. Moreover, we predicted the presence of mutational hotspots in the M. salmoniphilum isolates and 26% of these hotspots overlap with genes categorized as having roles in virulence, disease and defense. We also provide data about key genes involved in transcription and translation such as sigma factor, ribosomal protein and tRNA genes.

  • 2.
    Behra, Phani Rama Krishna
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Pettersson, Fredrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Das, Sarbashis
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Dasgupta, Santanu
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Kirsebom, Leif
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Comparative genomics of Mycobacterium mucogenicum and Mycobacterium neoaurum clade members emphasizing tRNA and non-coding RNA2019In: BMC Evolutionary Biology, ISSN 1471-2148, E-ISSN 1471-2148, Vol. 19, article id 124Article in journal (Refereed)
    Abstract [en]

    Background: Mycobacteria occupy various ecological niches and can be isolated from soil, tap water and ground water. Several cause diseases in humans and animals. To get deeper insight into our understanding of mycobacterial evolution focusing on tRNA and non-coding (nc)RNA, we conducted a comparative genome analysis of Mycobacterium mucogenicum (Mmuc) and Mycobacterium neoaurum (Mneo) clade members.

    Results: Genome sizes for Mmuc- and Mneo-clade members vary between 5.4 and 6.5 Mbps with the complete Mmuc(T) (type strain) genome encompassing 6.1 Mbp. The number of tRNA genes range between 46 and 79 (including one pseudo tRNA gene) with 39 tRNA genes common among the members of these clades, while additional tRNA genes were probably acquired through horizontal gene transfer. Selected tRNAs and ncRNAs (RNase P RNA, tmRNA, 4.5S RNA, Ms1 RNA and 6C RNA) are expressed, and the levels for several of these are higher in stationary phase compared to exponentially growing cells. The rare tRNA(Ile)TAT isoacceptor and two for mycobacteria novel ncRNAs: the Lactobacillales-derived GOLLD RNA and a homolog to the antisense Salmonella typhimurium phage Sar RNA, were shown to be present and expressed in certain Mmuc-clade members.

    Conclusions: Phages, IS elements, horizontally transferred tRNA gene clusters, and phage-derived ncRNAs appears to have influenced the evolution of the Mmuc- and Mneo-clades. While the number of predicted coding sequences correlates with genome size, the number of tRNA coding genes does not. The majority of the tRNA genes in mycobacteria are transcribed mainly from single genes and the levels of certain ncRNAs, including RNase P RNA (essential for the processing of tRNAs), are higher at stationary phase compared to exponentially growing cells. We provide supporting evidence that Ms1 RNA represents a mycobacterial 6S RNA variant. The evolutionary routes for the ncRNAs RNase P RNA, tmRNA and Ms1 RNA are different from that of the core genes.

  • 3.
    Caban, Kelvin
    et al.
    Columbia Univ, Dept Chem, New York, NY 10027 USA..
    Pavlov, Michael
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Kaledhonkar, Sandip
    Columbia Univ, Dept Biochem & Mol Biophys, New York, NY USA..
    Fu, Ziao
    Columbia Univ, Dept Biochem & Mol Biophys, New York, NY USA..
    Frank, Joachim
    Columbia Univ, Dept Biochem & Mol Biophys, New York, NY USA.;Columbia Univ, Dept Biol Sci, New York, NY 10027 USA..
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Gonzalez, Ruben L., Jr.
    Columbia Univ, Dept Chem, New York, NY 10027 USA..
    The Structural Basis for Initiation Factor 2 Activation during Translation Initiation2018In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 114, no 3, p. 593A-593AArticle in journal (Other academic)
  • 4.
    Choi, Junhong
    et al.
    Stanford Univ, Appl Phys, Stanford, CA 94305 USA..
    Indrisiunaite, Gabriele
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    DeMirci, Hasan
    SLAC Natl Accelerator Lab, Menlo Pk, CA USA..
    Ieong, Ka-Weng
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Wang, Jinfan
    Stanford Univ, Stanford, CA 94305 USA..
    Petrov, Alexey
    Stanford Univ, Stanford, CA 94305 USA..
    Prabhakar, Arjun
    Stanford Univ, Stanford, CA 94305 USA..
    Rechavi, Gideon
    Chaim Sheba Med Ctr, Canc Res Ctr, Tel Hashomer, Israel.;Tel Aviv Univ, Tel Aviv, Israel..
    Dominissini, Dan
    Tel Aviv Univ, Tel Aviv, Israel.;Chaim Sheba Med Ctr, Tel Hashomer, Israel..
    He, Chuan
    Univ Chicago, Chicago, IL 60637 USA..
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Puglisi, Joseph D.
    Stanford Univ, Stanford, CA 94305 USA..
    How 2 '-O-Methylation in mRNA Disrupts tRNA Decoding during Translation Elongation2018In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 114, no 3, p. 592A-592AArticle in journal (Other academic)
  • 5.
    Choi, Junhong
    et al.
    Stanford Univ, Sch Med, Dept Biol Struct, Stanford, CA 94305 USA.;Stanford Univ, Dept Appl Phys, Stanford, CA 94305 USA..
    Indrisiunaite, Gabriele
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    DeMirci, Hasan
    SLAC Natl Accelerator Lab, Stanford PULSE Inst, Menlo Pk, CA USA.;SLAC Natl Accelerator Lab, Biosci Div, Menlo Pk, CA USA..
    Leong, Ka-Weng
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Wang, Jinfan
    Stanford Univ, Sch Med, Dept Biol Struct, Stanford, CA 94305 USA..
    Petrov, Alexey
    Stanford Univ, Sch Med, Dept Biol Struct, Stanford, CA 94305 USA.;Auburn Univ, Dept Biol Sci, Auburn, AL 36849 USA..
    Prabhakarl, Arjun
    Stanford Univ, Sch Med, Dept Biol Struct, Stanford, CA 94305 USA.;Stanford Univ, Program Biophys, Stanford, CA 94305 USA..
    Rechavi, Gideon
    Chaim Sheba Med Ctr, Canc Res Ctr, Tel Hashomer, Israel.;Chaim Sheba Med Ctr, Wohl Ctr Translat Med, Tel Hashomer, Israel.;Tel Aviv Univ, Sackler Sch Med, Tel Aviv, Israel..
    Dominissini, Dan
    Chaim Sheba Med Ctr, Canc Res Ctr, Tel Hashomer, Israel.;Chaim Sheba Med Ctr, Wohl Ctr Translat Med, Tel Hashomer, Israel.;Tel Aviv Univ, Sackler Sch Med, Tel Aviv, Israel..
    He, Chuan
    Univ Chicago, Dept Biochem & Mol Biol, Dept Chem, 920 E 58Th St, Chicago, IL 60637 USA.;Univ Chicago, Inst Biophys Dynam, Chicago, IL 60637 USA.;Univ Chicago, Howard Hughes Med Inst, 5841 S Maryland Ave, Chicago, IL 60637 USA..
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Puglisi, Joseph D.
    Stanford Univ, Sch Med, Dept Biol Struct, Stanford, CA 94305 USA..
    2'-O-methylation in mRNA disrupts tRNA decoding during translation elongation2018In: Nature Structural & Molecular Biology, ISSN 1545-9993, E-ISSN 1545-9985, Vol. 25, no 3, p. 208-216Article in journal (Refereed)
    Abstract [en]

    Chemical modifications of mRNA may regulate many aspects of mRNA processing and protein synthesis. Recently, 2 '-O-methylation of nucleotides was identified as a frequent modification in translated regions of human mRNA, showing enrichment in codons for certain amino acids. Here, using single-molecule, bulk kinetics and structural methods, we show that 2 '-O-methylation within coding regions of mRNA disrupts key steps in codon reading during cognate tRNA selection. Our results suggest that 2 '-O-methylation sterically perturbs interactions of ribosomal-monitoring bases (G530, A1492 and A1493) with cognate codon-anticodon helices, thereby inhibiting downstream GTP hydrolysis by elongation factor Tu (EF-Tu) and A-site tRNA accommodation, leading to excessive rejection of cognate aminoacylated tRNAs in initial selection and proofreading. Our current and prior findings highlight how chemical modifications of mRNA tune the dynamics of protein synthesis at different steps of translation elongation.

  • 6.
    Cordeiro, Yraima
    et al.
    Univ Fed Rio de Janeiro, Fac Pharm, BR-21941902 Rio De Janeiro, Brazil.
    Vieira, Tuane
    Univ Fed Rio de Janeiro, Inst Bioquim Med Leopoldo Meis, Inst Nacl Ciencia Tecnol Biol Estrutural & Bioima, BR-21941902 Rio De Janeiro, Brazil.
    Kovachev, Petar Stefanov
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Silva, Jerson L.
    Univ Fed Rio de Janeiro, Inst Bioquim Med Leopoldo Meis, Inst Nacl Ciencia Tecnol Biol Estrutural & Bioima, BR-21941902 Rio De Janeiro, Brazil.
    Modulation of p53 and prion protein aggregation by RNA2019In: Biochimica et Biophysica Acta - Proteins and Proteomics, ISSN 1570-9639, E-ISSN 1878-1454, Vol. 1867, no 10, p. 933-940Article, review/survey (Refereed)
    Abstract [en]

    Several RNA-binding proteins undergo reversible liquid-liquid phase transitions, which, in pathological conditions, might evolve into transitions to solid-state phases, giving rise to amyloid structures. Amyloidogenic and prion-like proteins, such as the tumor suppressor protein p53 and the mammalian prion protein (PrP), bind RNAs specifically or nonspecifically, resulting in changes in their propensity to undergo aggregation. Mutant p53 aggregation seems to play a crucial role in cancer through loss of function, negative dominance and gain of function. PrP conversion modulated by RNA results in highly toxic aggregates. Here, we review data on the modulatory action of RNAs on the aggregation of both proteins.

  • 7.
    Das, Sarbashis
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Frisk, Christoffer
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Eriksson, Maria J.
    Karolinska Univ Hosp, Dept Clin Physiol, S-17176 Stockholm, Sweden;Karolinska Inst, Dept Mol Med & Surg, S-17177 Stockholm, Sweden.
    Walentinsson, Anna
    AstraZeneca, IMED Biotech Unit, Translat Sci Cardiovasc Renal & Metab Dis, S-43183 Gothenburg, Sweden.
    Corbascio, Matthias
    Karolinska Inst, Dept Mol Med & Surg, S-17177 Stockholm, Sweden;Karolinska Univ Hosp, Dept Thorac Surg, S-17176 Stockholm, Sweden.
    Hage, Camilla
    Karolinska Inst, Dept Med, S-17177 Stockholm, Sweden;Karolinska Univ Hosp, Heart & VascularTheme, S-17176 Stockholm, Sweden.
    Kumar, Chanchal
    AstraZeneca, IMED Biotech Unit, Translat Sci Cardiovasc Renal & Metab Dis, S-43183 Gothenburg, Sweden;Karolinska Inst, ICMC, Dept Med, S-14157 Huddinge, Sweden.
    Asp, Michaela
    Royal Inst Technol, Sci Life Lab, S-17121 Stockholm, Sweden.
    Lundeberg, Joakim
    Royal Inst Technol, Sci Life Lab, S-17121 Stockholm, Sweden.
    Maret, Eva
    Karolinska Univ Hosp, Dept Clin Physiol, S-17176 Stockholm, Sweden;Karolinska Inst, Dept Mol Med & Surg, S-17177 Stockholm, Sweden.
    Persson, Hans
    Karolinska Inst, Danderyd Hosp, Dept Clin Sci, S-18288 Stockholm, Sweden;Danderyd Hosp, Dept Cardiol, S-18288 Stockholm, Sweden.
    Linde, Cecilia
    Karolinska Inst, Dept Med, S-17177 Stockholm, Sweden;Karolinska Univ Hosp, Heart & VascularTheme, S-17176 Stockholm, Sweden.
    Persson, Bengt
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics. Karolinska Inst, Dept Med Biochem & Biophys, Sci Life Lab, S-17177 Stockholm, Sweden.
    Transcriptomics of cardiac biopsies reveals differences in patients with or without diagnostic parameters for heart failure with preserved ejection fraction2019In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 3179Article in journal (Refereed)
    Abstract [en]

    Heart failure affects 2-3% of adult Western population. Prevalence of heart failure with preserved left ventricular (LV) ejection fraction (HFpEF) increases. Studies suggest HFpEF patients to have altered myocardial structure and functional changes such as incomplete relaxation and increased cardiac stiffness. We hypothesised that patients undergoing elective coronary bypass surgery (CABG) with HFpEF characteristics would show distinctive gene expression compared to patients with normal LV physiology. Myocardial biopsies for mRNA expression analysis were obtained from sixteen patients with LV ejection fraction >= 45%. Five out of 16 patients (31%) had echocardiographic characteristics and increased NTproBNP levels indicative of HFpEF and this group was used as HFpEF proxy, while 11 patients had Normal LV physiology. Utilising principal component analysis, the gene expression data clustered into two groups, corresponding to HFpEF proxy and Normal physiology, and 743 differentially expressed genes were identified. The associated top biological functions were cardiac muscle contraction, oxidative phosphorylation, cellular remodelling and matrix organisation. Our results also indicate that upstream regulatory events, including inhibition of transcription factors STAT4, SRF and TP53, and activation of transcription repressors HEY2 and KDM5A, could provide explanatory mechanisms to observed gene expression differences and ultimately cardiac dysfunction in the HFpEF proxy group.

  • 8.
    Das, Sarbashis
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Pettersson, B. M. Fredrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Behra, Phani Rama Krishna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Mallick, Amrita
    Univ Louisiana, Dept Biol, Lafayette, LA USA.
    Cheramie, Martin
    Univ Louisiana, Dept Biol, Lafayette, LA USA.
    Ramesh, Malavika
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Shirreff, Lisa
    Univ Louisiana, Dept Biol, Lafayette, LA USA.
    DuCote, Tanner
    Univ Louisiana, Dept Biol, Lafayette, LA USA.
    Dasgupta, Santanu
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Ennis, Don G.
    Univ Louisiana, Dept Biol, Lafayette, LA USA.
    Kirsebom, Leif
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Extensive genomic diversity among Mycobacterium marinum strains revealed by whole genome sequencing2018In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, article id 12040Article in journal (Refereed)
    Abstract [en]

    Mycobacterium marinum is the causative agent for the tuberculosis-like disease mycobacteriosis in fish and skin lesions in humans. Ubiquitous in its geographical distribution, M. marinum is known to occupy diverse fish as hosts. However, information about its genomic diversity is limited. Here, we provide the genome sequences for 15 M. marinum strains isolated from infected humans and fish. Comparative genomic analysis of these and four available genomes of the M. marinum strains M, E11, MB2 and Europe reveal high genomic diversity among the strains, leading to the conclusion that M. marinum should be divided into two different clusters, the "M"- and the "Aronson"-type. We suggest that these two clusters should be considered to represent two M. marinum subspecies. Our data also show that the M. marinum pan-genome for both groups is open and expanding and we provide data showing high number of mutational hotspots in M. marinum relative to other mycobacteria such as Mycobacterium tuberculosis. This high genomic diversity might be related to the ability of M. marinum to occupy different ecological niches.

  • 9.
    Fislage, Marcus
    et al.
    VIB VUB Ctr Struct Biol, Brussels, Belgium;Columbia Univ, Dept Biochem & Mol Biophys, New York, NY 10027 USA;Vrije Univ Brussel, Struct Biol Brussels, Brussels, Belgium.
    Zhang, Jingji
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Columbia Univ, Dept Biochem & Mol Biophys, New York, NY 10027 USA.
    Brown, Zuben Patrick
    Osaka Univ, Inst Prot Res, Lab Prot Synth & Express, Osaka, Japan;Columbia Univ, Dept Biochem & Mol Biophys, New York, NY 10027 USA.
    Mandava, Chandra Sekhar
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Frank, Joachim
    Columbia Univ, Dept Biol Sci, New York, NY 10027 USA;Columbia Univ, Dept Biochem & Mol Biophys, New York, NY 10027 USA.
    Cryo-EM shows stages of initial codon selection on the ribosome by aa-tRNA in ternary complex with GTP and the GTPase-deficient EF-Tu(H84A)2018In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 46, no 11, p. 5861-5874Article in journal (Refereed)
    Abstract [en]

    The GTPase EF-Tu in ternary complex with GTP and aminoacyl-tRNA (aa-tRNA) promotes rapid and accurate delivery of cognate aa-tRNAs to the ribosomal A site. Here we used cryo-EM to study the molecular origins of the accuracy of ribosome-aided recognition of a cognate ternary complex and the accuracy-amplifying role of themonitoring bases A1492, A1493 and G530 of the 16S rRNA. We used the GTPase-deficient EF-Tu variant H84A with native GTP, rather than non-cleavable GTP analogues, to trap a near-cognate ternary complex in high-resolution ribosomal complexes of varying codon-recognition accuracy. We found that ribosome complexes trapped by GTPase-deficicent ternary complex due to the presence of EF-TuH84A or non-cleavable GTP analogues have very similar structures. We further discuss speed and accuracy of initial aa-tRNA selection in terms of conformational changes of aa-tRNA and stepwise activation of the monitoring bases at the decoding center of the ribosome.

  • 10.
    Fu, Ziao
    et al.
    Department of Biochemistry and Molecular Biophysics, Columbia University, New York, USA.
    Indrisiunaite, Gabriele
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Kaledhonkar, Sandip
    Department of Biochemistry and Molecular Biophysics, Columbia University, New York, USA.
    Shah, Binita
    Department of Biological Sciences, Barnard College, New York, USA.
    Sun, Ming
    Department of Biological Sciences, Colmbia University, New York, USA.
    Chen, Bo
    Department of Biological Sciences, Colmbia University, New York, USA.
    Grassucci, Robert A.
    Department of Biochemistry and Molecular Biophysics, Columbia University, New York, USA.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Frank, Joachim
    Department of Biochemistry and Molecular Biophysics, Columbia University, New York, USA; Department of Biological Sciences, Colmbia University, New York, USA.
    The structural basis for release-factor activation during translation termination revealed by time-resolved cryogenic electron microscopy2019In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 10, article id 2579Article in journal (Refereed)
    Abstract [en]

    When the ribosome encounters a stop codon, it recruits a release factor (RF) to hydrolyze the ester bond between the peptide chain and tRNA. RFs have structural motifs that recognize stop codons in the decoding center and a GGQ motif for induction of hydrolysis in the peptidyl transfer center 70 Å away. Surprisingly, free RF2 is compact, with only 20 Å between its codon-reading and GGQ motifs. Cryo-EM showed that ribosome-bound RFs have extended structures, suggesting that RFs are compact when entering the ribosome and then extend their structures upon stop codon recognition. Here we use time-resolved cryo-EM to visualize transient compact forms of RF1 and RF2 at 3.5 and 4 Å resolution, respectively, in the codon-recognizing ribosome complex on the native pathway. About 25% of complexes have RFs in the compact state at 24 ms reaction time, and within 60 ms virtually all ribosome-bound RFs are transformed to their extended forms.

  • 11.
    Ge, Xueliang
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Mandava, Chandra Sekhar
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Lind, Christoffer
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Complementary charge-based interaction between the ribosomal-stalk protein L7/12 and IF2 is the key to rapid subunit association2018In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 115, no 18, p. 4649-4654Article in journal (Refereed)
    Abstract [en]

    The interaction between the ribosomal-stalk protein L7/12 (L12) and initiation factor 2 (IF2) is essential for rapid subunit association, but the underlying mechanism is unknown. Here, we have characterized the L12–IF2 interaction on Escherichia coli ribosomes using site-directed mutagenesis, fast kinetics, and molecular dynamics (MD) simulations. Fifteen individual point mutations were introduced into the C-terminal domain of L12 (L12-CTD) at helices 4 and 5, which constitute the common interaction site for translational GTPases. In parallel, 15 point mutations were also introduced into IF2 between the G4 and G5 motifs, which we hypothesized as the potential L12 interaction sites. The L12 and IF2 mutants were tested in ribosomal subunit association assay in a stopped-flow instrument. Those amino acids that caused defective subunit association upon substitution were identified as the molecular determinants of L12–IF2 interaction. Further, MD simulations of IF2 docked onto the L12-CTD pinpointed the exact interacting partners—all of which were positively charged on L12 and negatively charged on IF2, connected by salt bridges. Lastly, we tested two pairs of charge-reversed mutants of L12 and IF2, which significantly restored the yield and the rate of formation of the 70S initiation complex. We conclude that complementary charge-based interaction between L12-CTD and IF2 is the key for fast subunit association. Considering the homology of the G domain, similar mechanisms may apply for L12 interactions with other translational GTPases.

  • 12.
    Ge, Xueliang
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Oliveira, Ana
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Hjort, Karin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Bergfors, Terese
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
    Gutiérrez-de-Terán, Hugo
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Andersson, Dan I
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Inhibition of translation termination by small molecules targeting ribosomal release factors2019In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 15424Article in journal (Refereed)
    Abstract [en]

    The bacterial ribosome is an important drug target for antibiotics that can inhibit different stages of protein synthesis. Among the various classes of compounds that impair translation there are, however, no known small-molecule inhibitors that specifically target ribosomal release factors (RFs). The class I RFs are essential for correct termination of translation and they differ considerably between bacteria and eukaryotes, making them potential targets for inhibiting bacterial protein synthesis. We carried out virtual screening of a large compound library against 3D structures of free and ribosome-bound RFs in order to search for small molecules that could potentially inhibit termination by binding to the RFs. Here, we report identification of two such compounds which are found both to bind free RFs in solution and to inhibit peptide release on the ribosome, without affecting peptide bond formation.

  • 13.
    Goronzy, I. N.
    et al.
    Stanford Univ, Dept Chem, Stanford, CA 94305 USA..
    Rawle, R. J.
    Univ Virginia, Dept Mol Physiol & Biomed Engn, Box 800886, Charlottesville, VA 22908 USA..
    Boxer, S. G.
    Stanford Univ, Dept Chem, Stanford, CA 94305 USA..
    Kasson, Peter M.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology. Univ Virginia, Dept Mol Physiol & Biomed Engn, Box 800886, Charlottesville, VA 22908 USA..
    Cholesterol enhances influenza binding avidity by controlling nanoscale receptor clustering2018In: Chemical Science, ISSN 2041-6520, E-ISSN 2041-6539, Vol. 9, no 8, p. 2340-2347Article in journal (Refereed)
    Abstract [en]

    Influenza virus infects cells by binding to sialylated glycans on the cell surface. While the chemical structure of these glycans determines hemagglutinin-glycan binding affinity, bimolecular affinities are weak, so binding is avidity-dominated and driven by multivalent interactions. Here, we show that membrane spatial organization can control viral binding. Using single-virus fluorescence microscopy, we demonstrate that the sterol composition of the target membrane enhances viral binding avidity in a dose-dependent manner. Binding shows a cooperative dependence on concentration of receptors for influenza virus, as would be expected for a multivalent interaction. Surprisingly, the ability of sterols to promote viral binding is independent of their ability to support liquid-liquid phase separation in model systems. We develop a molecular explanation for this observation via molecular dynamics simulations, where we find that cholesterol promotes small-scale clusters of glycosphingolipid receptors. We propose a model whereby cholesterol orders the monomeric state of glycosphingolipid receptors, reducing the entropic penalty of receptor association and thus favoring multimeric complexes without phase separation. This model explains how cholesterol and other sterols control the spatial organization of membrane receptors for influenza and increase viral binding avidity. A natural consequence of this finding is that local cholesterol concentration in the plasma membrane of cells may alter the binding avidity of influenza virions. Furthermore, our results demonstrate a form of cholesterol-dependent membrane organization that does not involve lipid rafts, suggesting that cholesterol's effect on cell membrane heterogeneity is likely the interplay of several different factors.

  • 14.
    Harish, Ajith
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology. Uppsala Univ, Dept Cell & Mol Biol, Program Mol Biol, Uppsala, Sweden.
    What is an archaeon and are the Archaea really unique?2018In: PeerJ, ISSN 2167-8359, E-ISSN 2167-8359, Vol. 6, article id e5770Article in journal (Refereed)
    Abstract [en]

    The recognition of the group Archaea as a major branch of the tree of life (ToL) prompted a new view of the evolution of biodiversity. The genomic representation of archaeal biodiversity has since significantly increased. In addition, advances in phylogenetic modeling of multi-locus datasets have resolved many recalcitrant branches of the ToL. Despite the technical advances and an expanded taxonomic representation, two important aspects of the origins and evolution of the Archaea remain controversial, even as we celebrate the 40th anniversary of the monumental discovery. These issues concern (i) the uniqueness (monophyly) of the Archaea, and (ii) the evolutionary relationships of the Archaea to the Bacteria and the Eukarya; both of these are relevant to the deep structure of the ToL. To explore the causes for this persistent ambiguity, I examine multiple datasets and different phylogenetic approaches that support contradicting conclusions. I find that the uncertainty is primarily due to a scarcity of information in standard datasets-universal core-genes datasets-to reliably resolve the conflicts. These conflicts can be resolved efficiently by comparing patterns of variation in the distribution of functional genomic signatures, which are less diffused unlike patterns of primary sequence variation. Relatively lower heterogeneity in distribution patterns minimizes uncertainties and supports statistically robust phylogenetic inferences, especially of the earliest divergences of life. This case study further highlights the limitations of primary sequence data in resolving difficult phylogenetic problems, and raises questions about evolutionary inferences drawn from the analyses of sequence alignments of a small set of core genes. In particular, the findings of this study corroborate the growing consensus that reversible substitution mutations may not be optimal phylogenetic markers for resolving early divergences in the ToL, nor for determining the polarity of evolutionary transitions across the ToL.

  • 15.
    Holm, Mikael
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Mandava, Chandra Sekhar
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    The mechanism of error induction by the antibiotic viomycin provides insight into the fidelity mechanism of translation2019In: eLIFE, E-ISSN 2050-084X, Vol. 8, article id e46124Article in journal (Refereed)
    Abstract [en]

    Applying pre-steady state kinetics to an Escherichia-coli-based reconstituted translation system, we have studied how the antibiotic viomycin affects the accuracy of genetic code reading. We find that viomycin binds to translating ribosomes associated with a ternary complex (TC) consisting of elongation factor Tu (EF-Tu), aminoacyl tRNA and GTP, and locks the otherwise dynamically flipping monitoring bases A1492 and A1493 into their active conformation. This effectively prevents dissociation of near- and non-cognate TCs from the ribosome, thereby enhancing errors in initial selection. Moreover, viomycin shuts down proofreading-based error correction. Our results imply a mechanism in which the accuracy of initial selection is achieved by larger backward rate constants toward TC dissociation rather than by a smaller rate constant for GTP hydrolysis for near- and non-cognate TCs. Additionally, our results demonstrate that translocation inhibition, rather than error induction, is the major cause of cell growth inhibition by viomycin.

  • 16.
    Ieong, Ka-Weng
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology. Institute for Medical Engineering & Science, Department of Biological Engineering, and Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, MA, USA. Infectious Disease and Microbiome Program, Broad Institute, Cambridge, MA, USA.
    Indrisiunaite, Gabriele
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    N6-methyladenosines in mRNA have profound effects on the accuracy of codon reading by tRNAs and peptide release factorsManuscript (preprint) (Other academic)
  • 17.
    Indrisiunaite, Gabriele
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Accuracy of protein synthesis and its tuning by mRNA modifications2019Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The ribosome is a large macromolecular complex that synthesizes all proteins in the cell in all kingdoms of life. Proteins perform many vital functions, ranging from catalysis of biochemical reactions to muscle movement. It is essential for cells and organisms that proteins are synthesized rapidly and accurately.

    This thesis addresses two questions regarding the accuracy of protein synthesis. How do bacterial and eukaryotic release factors ensure accurate termination? How do mRNA modifications affect the accuracy of bacterial protein synthesis?

    Bacterial release factors 1 (RF1) and 2 (RF2) are proteins that recognize the stop codons of mRNA and catalyze the release of a synthesized protein chain from the ribosome. It has been proposed that RFs ensure accurate termination by binding to the ribosome in an inactive, compact conformation and acquire a catalytically active, extended conformation only after recognizing a correct stop codon. However, the native compact conformation was too short-lived to be captured by conventional structural methods. We have developed a fast-kinetics approach for determining when the RFs are in a compact conformation on the ribosome and then used time-resolved cryogenic electron microscopy to capture the compact conformations of native RF1 and RF2 bound to a stop codon. We have also measured the effect of eukaryotic release factor 3 (eRF3) on the rate and accuracy of peptide release by eukaryotic release factor 1 (eRF1) in a yeast (Saccharomyces cerevisiae) in vitro translation system.

    Modifications of mRNA nucleotides are post-transcriptional regulators of gene expression, but little is known about their role in protein synthesis. We have studied the effect on accuracy of protein synthesis by two of these modifications: 2’-O-methylation and N6-methylation of adenosine. 2’-O-methylation greatly reduced the maximal rate (kcat) and efficiency (kcat/Km) of cognate (correct) codon reading by decreasing the initial GTPase activity in elongation factor Tu and enhancing proofreading losses of cognate aminoacyl-tRNAs. Remarkably, N6-methylation reduced the efficiency of codon reading by cognate aminoacyl-tRNAs and release factors, leaving the efficiency of the corresponding non-cognate reactions much less affected.

     

  • 18.
    Kipper, Kalle
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Eremina, Nadja
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Marklund, Emil
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Tubasum, Sumera
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Mao, Guanzhong
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lehmann, Laura Christina
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Elf, Johan
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Deindl, Sebastian
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Structure-guided approach to site-specific fluorophore labeling of the lac repressor LacI2018In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 13, no 6, article id e0198416Article in journal (Refereed)
    Abstract [en]

    The lactose operon repressor protein LacI has long served as a paradigm of the bacterial transcription factors. However, the mechanisms whereby LacI rapidly locates its cognate binding site on the bacterial chromosome are still elusive. Single-molecule fluorescence imaging approaches are well suited for the study of these mechanisms but rely on a functionally compatible fluorescence labeling of LacI. Particularly attractive for protein fluorescence labeling are synthetic fluorophores due to their small size and favorable photophysical characteristics. Synthetic fluorophores are often conjugated to natively occurring cysteine residues using maleimide chemistry. For a site-specific and functionally compatible labeling with maleimide fluorophores, the target protein often needs to be redesigned to remove unwanted native cysteines and to introduce cysteines at locations better suited for fluorophore attachment. Biochemical screens can then be employed to probe for the functional activity of the redesigned protein both before and after dye labeling. Here, we report a mutagenesis- based redesign of LacI to enable a functionally compatible labeling with maleimide fluorophores. To provide an easily accessible labeling site in LacI, we introduced a single cysteine residue at position 28 in the DNA-binding headpiece of LacI and replaced two native cysteines with alanines where derivatization with bulky substituents is known to compromise the protein's activity. We find that the redesigned LacI retains a robust activity in vitro and in vivo, provided that the third native cysteine at position 281 is retained in LacI. In a total internal reflection microscopy assay, we observed individual Cy3-labeled LacI molecules bound to immobilized DNA harboring the cognate O-1 operator sequence, indicating that the dye-labeled LacI is functionally active. We have thus been able to generate a functional fluorescently labeled LacI that can be used to unravel mechanistic details of LacI target search at the single molecule level.

  • 19.
    Koshla, Oksana
    et al.
    Ivan Franko Natl Univ Lviv, Dept Genet & Biotechnol, 4 Hrushevskoho St, UA-79005 Lvov, Ukraine.
    Yushchuk, Oleksandr
    Ivan Franko Natl Univ Lviv, Dept Genet & Biotechnol, 4 Hrushevskoho St, UA-79005 Lvov, Ukraine.
    Stash, Iryna
    Ivan Franko Natl Univ Lviv, Dept Genet & Biotechnol, 4 Hrushevskoho St, UA-79005 Lvov, Ukraine.
    Dacyuk, Yuriy
    Ivan Franko Natl Univ Lviv, Dept Phys Earth, 4 Hrushevskoho St, UA-79005 Lvov, Ukraine.
    Myronovskyi, Maksym
    Helmholtz Inst Pharmaceut Res, Saarland Campus,Bldg C2-3, D-66123 Saarbrucken, Germany.
    Jager, Gunilla
    Umea Univ, Dept Mol Biol, 6K Och 6L, S-90197 Umea, Sweden.
    Sussmuth, Roderich D.
    Tech Univ Berlin, Inst Chem, Str 17 Juni 124-TC2, D-10623 Berlin, Germany.
    Luzhetskyy, Andriy
    Helmholtz Inst Pharmaceut Res, Saarland Campus,Bldg C2-3, D-66123 Saarbrucken, Germany.
    Bystrom, Anders
    Umea Univ, Dept Mol Biol, 6K Och 6L, S-90197 Umea, Sweden.
    Kirsebom, Leif
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Ostash, Bohdan
    Ivan Franko Natl Univ Lviv, Dept Genet & Biotechnol, 4 Hrushevskoho St, UA-79005 Lvov, Ukraine.
    Gene miaA for post-transcriptional modification of tRNAXXA is important for morphological and metabolic differentiation in Streptomyces2019In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 112, no 1, p. 249-265Article in journal (Refereed)
    Abstract [en]

    Members of actinobacterial genus Streptomyces possess a sophisticated life cycle and are the deepest source of bioactive secondary metabolites. Although morphogenesis and secondary metabolism are subject to transcriptional co-regulation, streptomycetes employ an additional mechanism to initiate the aforementioned processes. This mechanism is based on delayed translation of rare leucyl codon UUA by the only cognate tRNA(UAA)(Leu) (encoded by bldA). The bldA-based genetic switch is an extensively documented example of translational regulation in Streptomyces. Yet, after five decades since the discovery of bldA, factors that shape its function and peculiar conditionality remained elusive. Here we address the hypothesis that post-transcriptional tRNA modifications play a role in tRNA-based mechanisms of translational control in Streptomyces. Particularly, we studied two Streptomyces albus J1074 genes, XNR_1074 (miaA) and XNR_1078 (miaB), encoding tRNA (adenosine(37)-N6)-dimethylallyltransferase and tRNA (N6-isopentenyl adenosine(37)-C2)-methylthiotransferase respectively. These enzymes produce, in a sequential manner, a hypermodified ms(2)i(6)A37 residue in most of the A36-A37-containing tRNAs. We show that miaB and especially miaA null mutant of S. albus possess altered morphogenesis and secondary metabolism. We provide genetic evidence that miaA deficiency impacts translational level of gene expression, most likely through impaired decoding of codons UXX and UUA in particular.

  • 20.
    Liljas, Anders
    et al.
    Lund Univ, Dept Biochem & Struct Biol, Ctr Chem & Chem Engn, Lund, Sweden.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    The enigmatic ribosomal stalk2018In: Quarterly reviews of biophysics (Print), ISSN 0033-5835, E-ISSN 1469-8994, Vol. 51, article id e12Article, review/survey (Refereed)
    Abstract [en]

    The large ribosomal subunit has a distinct feature, the stalk, extending outside the ribosome. In bacteria it is called the L12 stalk. The base of the stalk is protein uL10 to which two or three dimers of proteins bL12 bind. In archea and eukarya P1 and P2 proteins constitute the stalk. All these extending proteins, that have a high degree of flexibility due to a hinge between their N- and C-terminal parts, are essential for proper functionalization of some of the translation factors. The role of the stalk proteins has remained enigmatic for decades but is gradually approaching an understanding. In this review we summarise the knowhow about the structure and function of the ribosomal stalk till date starting from the early phase of ribosome research.

  • 21.
    Liljeruhm, Josefine
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Exotic Ribosomal Enzymology2019Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    This thesis clarifies intriguing enzymology of the ribosome, the multiRNA/multiprotein complex that catalyzes protein synthesis (translation). The large ribosomal RNAs (23S and 16S rRNAs in E. coli) are post-transcriptionally modified by many specific modification enzymes, yet the functions of the modifications remain enigmatic. A deeper insight into two of the 23S rRNA S-adenosyl-methionine-requiring methyltransferase enzymes, RlmM and RlmJ, was given by investigating substrate specificity in vitro. Both enzymes were able to methylate in vitro-transcribed, modification-free, protein-free, 2659-nucleotide-long 23S rRNA. Furthermore, RlmM was able to methylate the 611-nucleotide-long Domain V of the 23S rRNA alone and RlmJ could modify the A2030 with only 25 surrounding nucleotides.

    Translation is evolutionary optimized to incorporate L-amino acids to the exclusion of D-amino acids in the cell. To understand how, and how to engineer around this restriction for pharmacological applications, detailed kinetics of ribosomal dipeptide formation with D- versus L-phenylalanine-tRNA were determined. This was done by varying the concentrations of EF-Tu (which delivers aminoacyl-tRNAs to the ribosome) and the ribosome, as well as changing the tRNA adaptor. Binding to EF-Tu was shown to be rate limiting for D-Phe-tRNA at a low concentration of EF-Tu. Surprisingly, at a higher (physiological) concentration of EF-Tu, binding and subsequent dipeptide synthesis became so efficient that D-Phe incorporation became competitive with L-Phe, and accommodation/peptide bond formation was unmasked as a new rate-limiting step. This highlighted the importance of D-aminoacyl-tRNA deacylase in restricting translation with D-amino acids in vivo.

    Although polypeptides are intrinsically colorless, it is remarkable that evolution has nevertheless enabled ribosomes to synthesize highly colored proteins (chromoproteins). Such eukaryotic proteins reside in coral reefs and undergo self-catalyzed, intramolecular, chromophore formation by reacting with oxygen in a manner highly similar to that of green fluorescent protein. The potential utility of different colored chromoproteins in E. coli was analyzed via codon-optimized over-expression and quantification of maturation times, color intensities and cellular fitness costs. No chromoprotein was found to have the combined characteristics of fast maturation, intense color and low fitness cost. However, semi-rational mutagenesis created different colored variants with identical fitness costs suitable for competition assays and teaching.

  • 22.
    Liljeruhm, Josefine
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Funk, Saskia K.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Tietscher, Sandra
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Edlund, Anders D.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala Univ, iGEM Uppsala, Uppsala, Sweden.
    Jamal, Sabri
    Uppsala Univ, iGEM Uppsala, Uppsala, Sweden.
    Yuen, Pikkei
    Uppsala Univ, iGEM Uppsala, Uppsala, Sweden.
    Dyrhage, Karl
    Uppsala Univ, iGEM Uppsala, Uppsala, Sweden.
    Gynnå, Arvid H.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala Univ, iGEM Uppsala, Uppsala, Sweden.
    Ivermark, Katarina
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    Lövgren, Jessica
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    Törnblom, Viktor
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    Virtanen, Anders
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Lundin, Erik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala Univ, iGEM Uppsala, Uppsala, Sweden.
    Wistand-Yuen, Erik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Forster, Anthony C.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Engineering a palette of eukaryotic chromoproteins for bacterial synthetic biology2018In: Journal of Biological Engineering, ISSN 1754-1611, E-ISSN 1754-1611, Vol. 12, article id 8Article in journal (Refereed)
    Abstract [en]

    Background: Coral reefs are colored by eukaryotic chromoproteins (CPs) that are homologous to green fluorescent protein. CPs differ from fluorescent proteins (FPs) by intensely absorbing visible light to give strong colors in ambient light. This endows CPs with certain advantages over FPs, such as instrument-free detection uncomplicated by ultra-violet light damage or background fluorescence, efficient Forster resonance energy transfer (FRET) quenching, and photoacoustic imaging. Thus, CPs have found utility as genetic markers and in teaching, and are attractive for potential cell biosensor applications in the field. Most near-term applications of CPs require expression in a different domain of life: bacteria. However, it is unclear which of the eukaryotic CP genes might be suitable and how best to assay them.

    Results: Here, taking advantage of codon optimization programs in 12 cases, we engineered 14 CP sequences (meffRed, eforRed, asPink, spisPink, scOrange, fwYellow, amilGFP, amajLime, cjBlue, mefiBlue, aeBlue, amilCP, tsPurple and gfasPurple) into a palette of Escherichia coil BioBrick plasmids. BioBricks comply with synthetic biology's most widely used, simplified, cloning standard. Differences in color intensities, maturation times and fitness costs of expression were compared under the same conditions, and visible readout of gene expression was quantitated. A surprisingly large variation in cellular fitness costs was found, resulting in loss of color in some overnight liquid cultures of certain high-copy-plasmid-borne CPs, and cautioning the use of multiple CPs as markers in competition assays. We solved these two problems by integrating pairs of these genes into the chromosome and by engineering versions of the same CP with very different colors.

    Conclusion: Availability of 14 engineered CP genes compared in E coil, together with chromosomal mutants suitable for competition assays, should simplify and expand CP study and applications. There was no single plasmid-borne CP that combined all of the most desirable features of intense color, fast maturation and low fitness cost, so this study should help direct future engineering efforts.

  • 23.
    Liljeruhm, Josefine
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Wang, Jinfan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Kwiatkowski, Marek
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Sabari, Samudra
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Forster, Anthony C.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Kinetics of D-amino acid incorporation in translation2019In: ACS Chemical Biology, ISSN 1554-8929, E-ISSN 1554-8937, Vol. 14, no 2, p. 204-213Article in journal (Refereed)
    Abstract [en]

    Despite the stereospecificity of translation for l-amino acids (l-AAs) in vivo, synthetic biologists have enabled ribosomal incorporation of d-AAs in vitro toward encoding polypeptides with pharmacologically desirable properties. However, the steps in translation limiting d-AA incorporation need clarification. In this work, we compared d- and l-Phe incorporation in translation by quench-flow kinetics, measuring 250-fold slower incorporation into the dipeptide for the d isomer from a tRNAPhe-based adaptor (tRNAPheB). Incorporation was moderately hastened by tRNA body swaps and higher EF-Tu concentrations, indicating that binding by EF-Tu can be rate-limiting. However, from tRNAAlaB with a saturating concentration of EF-Tu, the slow d-Phe incorporation was unexpectedly very efficient in competition with incorporation of the l isomer, indicating fast binding to EF-Tu, fast binding of the resulting complex to the ribosome, and rate-limiting accommodation/peptide bond formation. Subsequent elongation with an l-AA was confirmed to be very slow and inefficient. This understanding helps rationalize incorporation efficiencies in vitro and stereospecific mechanisms in vivo and suggests approaches for improving incorporation.

  • 24.
    Lindström, John
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Method for tracking orthogonal ribosomes in vivo using MS2coat protein2019Independent thesis Advanced level (professional degree), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Ribosomes are large macromolecules responsible for protein synthesisand they consist of both RNA and proteins. Each ribosome is made of one large and one small subunit. Even though the ribosome is one of the most studied machineries in the cell there is a gap in our understanding of how this macromolecule functions in vivo. In this project we aimed to develop a method for tracking a specific subset of ribosomes using super-resolution fluorescence microscopy. This was achieved by using the MS2 coat protein (MS2CP) fused to a fluorescent marker and by modifying ribosomes to have the RNA loop to which theMS2CP binds with high affinity. We were able to obtain the most promising results when the MS2CP was fused to a Halotag with the dye JF549 attached to it. The JF549 has good cell permeability which allows simple and efficient labelling of ribosomes. To be able to observe translation of specific mRNAs we used this labelling strategy to track orthogonal ribosomes which do not recognise mRNA normally produced in cells but can translate mRNAs with a modified 5’-UTR.Orthogonal ribosomes were tested with several different 5´-UTRs. With the construct for which we obtained the highest expression level we observed that up to 43% of the labelled orthogonal ribosomes were engaged in translation of the specific mRNA. The system will make it possible to determine how the sequence of a particular mRNA will affect its in vivo translation.

  • 25.
    Mao, Guanzhong
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Srivastava, Abhishek S.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology. AstraZeneca R&D, Discovery Sci, Cambridge Sci Pk, Cambridge, England..
    Wu, Shiying
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Kosek, David
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Lindell, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Kirsebom, Leif
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Critical domain interactions for type A RNase P RNA catalysis with and without the specificity domain2018In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 13, no 3, article id e0192873Article in journal (Refereed)
    Abstract [en]

    The natural trans-acting ribozyme RNase P RNA (RPR) is composed of two domains in which the catalytic (C-) domain mediates cleavage of various substrates. The C-domain alone, after removal of the second specificity (S-) domain, catalyzes this reaction as well, albeit with reduced efficiency. Here we provide experimental evidence indicating that efficient cleavage mediated by the Escherichia coli C-domain (Eco CP RPR) with and without the C5 protein likely depends on an interaction referred to as the "P6-mimic". Moreover, the P18 helix connects the C-and S-domains between its loop and the P8 helix in the S-domain (the P8/P18-interaction). In contrast to the "P6-mimic", the presence of P18 does not contribute to the catalytic performance by the C-domain lacking the S-domain in cleavage of an all ribo model hairpin loop substrate while deletion or disruption of the P8/P18-interaction in full-size RPR lowers the catalytic efficiency in cleavage of the same model hairpin loop substrate in keeping with previously reported data using precursor tRNAs. Consistent with that P18 is not required for cleavage mediated by the C-domain we show that the archaeal Pyrococcus furiosus RPR C-domain, which lacks the P18 helix, is catalytically active in trans without the S-domain and any protein. Our data also suggest that the S-domain has a larger impact on catalysis for E. coli RPR compared to P. furiosus RPR. Finally, we provide data indicating that the absence of the S-domain and P18, or the P8/P18-interaction in full-length RPR influences the charge distribution near the cleavage site in the RPR-substrate complex to a small but reproducible extent.

  • 26.
    Mellenius, Harriet
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Transcriptional accuracy modeling suggests two-step proofreading by RNA polymerase2017In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 45, no 20, p. 11582-11593Article in journal (Refereed)
    Abstract [en]

    We suggest a novel two-step proofreading mechanism with two sequential rounds of proofreading selection in mRNA transcription. It is based on the previous experimental observations that the proofreading RNA polymerase cleaves off transcript fragments of at least 2 nt and that transcript elongation after a nucleotide misincorporation is anomalously slow. Taking these results into account, we extend the description of the accuracy of template guided nucleotide selection beyond previous models of RNA polymerase-dependent DNA transcription. The model derives the accuracy of initial and proofreading base selection from experimentally estimated nearest-neighbor parameters. It is also used to estimate the small accuracy enhancement of polymerase revisiting of previous positions following transcript cleavage.

  • 27.
    Mishra, Abhishek Kumar
    et al.
    Ctr Biomed Res CBMR, Div Mol Synth & Drug Discovery, SGPGIMS Campus, Lucknow 226014, Uttar Pradesh, India.
    Morgon, Nelson Henrique
    Univ Estadual Campinas, UNICAMP, Inst Chem, Dept Phys Chem, BR-13083970 Campinas, SP, Brazil.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    de Souza, Aguinaldo Robinson
    Sao Paulo State Univ, UNESP, Sch Sci, BR-17033360 Bauru, SP, Brazil.
    Biswas, Srijit
    Ctr Biomed Res CBMR, Div Mol Synth & Drug Discovery, SGPGIMS Campus, Lucknow 226014, Uttar Pradesh, India;Univ Calcutta, Rajabazar Sci Coll Campus, Dept Chem, Kolkata 700009, W Bengal, India.
    Catalytic O- to N-Alkyl Migratory Rearrangement: Transition Metal-Free Direct and Tandem Routes to N-Alkylated Pyridones and Benzothiazolones2018In: Advanced Synthesis and Catalysis, ISSN 1615-4150, E-ISSN 1615-4169, Vol. 360, no 20, p. 3930-3939Article in journal (Refereed)
    Abstract [en]

    The present study reports the synthesis of N-alkylated pyridones and benzothiazolones via O- to N-alkyl group migration under transition metal-free TfOH-catalyzed reaction conditions for the first time, to the best of our knowledge. Primary as well as secondary alkyl groups smoothly migrate under the present reaction conditions. Moreover, a minor modification of the protocol used in this study is found to be applicable for an entirely new tandem synthesis of 2-alkoxy-N-heterocycles from the simplest starting materials in a solvent-free reaction conditions. Density Functional Theory (DFT) calculation identifies the energy species associated with the rearrangement, whereas, mechanistic experiments explore the role of the catalyst as the alkyl group transfer mediator.

  • 28.
    Moriou, Céline
    et al.
    CNRS, Inst Chim Subst Nat, Gif Sur Yvette, France.
    Da Silva, Adilson D.
    Univ Fed Juiz de Fora, Dept Quim, ICE, BR-Juiz De Fora, MG, Brazil.
    Vianelli Prado, Marcos Joel
    Univ Fed Juiz de Fora, Dept Quim, ICE, BR-Juiz De Fora, MG, Brazil.
    Denhez, Clément
    Univ Reims, Inst Chim Mol Reims, CNRS, UMR 7312,UFR Pharm, 51 Rue Cognacq Jay, Reims, France; Univ Reims, Multiscale Mol Modelling Platform, UFR Sci Exactes & Nat, Reims, France.
    Plashkevych, Oleksandr
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Chattopadhyaya, Jyoti
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Guillaume, Dominique
    Univ Reims, Inst Chim Mol Reims, CNRS, UMR 7312,UFR Pharm, 51 Rue Cognacq Jay, Reims, France.
    Clivio, Pascale
    Univ Reims, Inst Chim Mol Reims, CNRS, UMR 7312, UFR Pharm, 51 Rue Cognacq Jay, Reims, France.
    C2 '-F Stereoconfiguration As a Puckering Switch for Base Stacking at the Dinucleotide Level2018In: Journal of Organic Chemistry, ISSN 0022-3263, E-ISSN 1520-6904, Vol. 83, no 4, p. 2473-2478Article in journal (Refereed)
    Abstract [en]

    Fluorine configuration at C2′ of the bis(2′-fluorothymidine) dinucleotide is demonstrated to drive intramolecular base stacking. 2′-β F-Configuration drastically reduces stacking compared to the 2′-α series. Hence, base stacking emerges as being tunable by the C2′-F stereoconfiguration through dramatic puckering variations scrutinized by NMR and natural bond orbital analysis. Accordingly, 2′-β F-isomer photoreactivity is significantly reduced compared to that of the 2′-α F-isomer.

  • 29.
    Nissbeck, Mikael
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Emmerich, Andrew
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Virtanen, Anders
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Mechanisms of PARN action: Kinetic analysis of processive mode of degradationManuscript (preprint) (Other academic)
  • 30.
    Pavlov, Michael
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Substrate-Induced Formation of Ribosomal Decoding Center for Accurate and Rapid Genetic Code Translation2018In: Annual Review of Biophysics, ISSN 1936-122X, E-ISSN 1936-1238, p. 525-548Article, review/survey (Refereed)
    Abstract [en]

    Accurate translation of genetic information is crucial for synthesis of functional proteins in all organisms. We use recent experimental data to discuss how induced fit affects accuracy of initial codon selection on the ribosome by aminoacyl transfer RNA in ternary complex (T-3) with elongation factor Tu (EF-Tu) and guanosine-5'-triphosphate (GTP). We define actual accuracy (A(I)(nc)) of a particular protein synthesis system as its current accuracy and the effective selectivity (d(eI)(nc)) as A(I)(nc) in the limit of zero ribosomal binding affinity for T-3. Intrinsic selectivity (D-I(nc)), defined as the upper thermodynamic limit of d(eI)(nc), is determined by the free energy difference between near-cognate and cognate T-3 in the pre-GTP hydrolysis state on the ribosome. D-I(nc) is much larger than d(eI)(nc), suggesting the possibility of a considerable increase in d(eI)(nc) and A(I)(nc) at negligible kinetic cost. Induced fit increases A(I)(nc) and d(eI)(nc) without affecting D-I(nc), and aminoglycoside antibiotics reduce A(I)(nc) and d(eI)(nc) at unaltered D-I(nc).

  • 31.
    Togtema, Melissa
    et al.
    Thunder Bay Reg Hlth Res Inst, Probe Dev & Biomarker Explorat, Thunder Bay, ON, Canada; Lakehead Univ, Biotechnol Program, Thunder Bay, ON, Canada.
    Jackson, Robert
    Thunder Bay Reg Hlth Res Inst, Probe Dev & Biomarker Explorat, Thunder Bay, ON, Canada; Lakehead Univ, Biotechnol Program, Thunder Bay, ON, Canada.
    Grochowski, Jessica
    Thunder Bay Reg Hlth Res Inst, Probe Dev & Biomarker Explorat, Thunder Bay, ON, Canada; UHN, Michener Inst Educ, Genet Technol Program, Toronto, ON, Canada.
    Villa, Peter L.
    Thunder Bay Reg Hlth Res Inst, Probe Dev & Biomarker Explorat, Thunder Bay, ON, Canada; Lakehead Univ, Dept Biol, Thunder Bay, ON, Canada.
    Mellerup, Miranda
    Thunder Bay Reg Hlth Res Inst, Probe Dev & Biomarker Explorat, Thunder Bay, ON, Canada; Univ Ottawa, Dept Biol, Ottawa, ON, Canada.
    Chattopadhyaya, Jyoti
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Zehbe, Ingeborg
    Thunder Bay Reg Hlth Res Inst, Probe Dev & Biomarker Explorat, Thunder Bay, ON, Canada; Lakehead Univ, Dept Biol, Thunder Bay, ON, Canada.
    Synthetic siRNA targeting human papillomavirus 16 E6: a perspective on in vitro nanotherapeutic approaches2018In: Nanomedicine, ISSN 1743-5889, E-ISSN 1748-6963, Vol. 13, no 4, p. 455-474Article in journal (Other academic)
    Abstract [en]

    High-risk human papillomaviruses infect skin and mucosa, causing approximately 5% of cancers worldwide. In the search for targeted nanotherapeutic approaches, siRNAs against the viral E6 transcript have been molecules of interest but have not yet seen successful translation into the clinic. By reviewing the past approximately 15 years of in vitro literature, we identify the need for siRNA validation protocols which concurrently evaluate ranges of key treatment parameters as well as characterize downstream process restoration in a methodical, quantitative manner and demonstrate their implementation using our own data. We also reflect on the future need for more appropriate cell culture models to represent patient lesions as well as the application of personalized approaches to identify optimal treatment strategies.

  • 32.
    Volkov, Ivan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Lindén, Martin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Aguirre, Javier
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Ieong, Ka-Weng
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Metelev, Mikhail
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Johansson, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    tRNA tracking for direct measurements of protein synthesis kinetics in live cells2018In: Nature Chemical Biology, ISSN 1552-4450, E-ISSN 1552-4469, Vol. 14, no 6, p. 618-626Article in journal (Refereed)
    Abstract [en]

    Our ability to directly relate results from test-tube biochemical experiments to the kinetics in living cells is very limited. Here we present experimental and analytical tools to directly study the kinetics of fast biochemical reactions in live cells. Dye-labeled molecules are electroporated into bacterial cells and tracked using super-resolved single-molecule microscopy.Trajectories are analyzed by machine-learning algorithms to directly monitor transitions between bound and free states. In particular, we measure the dwell time of tRNAs on ribosomes, and hence achieve direct measurements of translation rates inside living cells at codon resolution. We find elongation rates with tRNA(Phe) that are in perfect agreement with previous indirect estimates, and once fMet-tRNA(fMet) has bound to the 30S ribosomal subunit, initiation of translation is surprisingly fast and does not limit the overall rate of protein synthesis. The experimental and analytical tools for direct kinetics measurements in live cells have applications far beyond bacterial protein synthesis.

  • 33.
    Zhang, Jingji
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Pavlov, Michael
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Accuracy of genetic code translation and its orthogonal corruption by aminoglycosides and Mg2+ ions2018In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 46, no 3, p. 1362-1374Article in journal (Refereed)
    Abstract [en]

    We studied the effects of aminoglycosides and changing Mg2+ ion concentration on the accuracy of initial codon selection by aminoacyl-tRNA in ternary complex with elongation factor Tu and GTP (T-3) on mRNA programmed ribosomes. Aminoglycosides decrease the accuracy by changing the equilibrium constants of 'monitoring bases' A1492, A1493 and G530 in 16S rRNA in favor of their 'activated' state by large, aminoglycoside-specific factors, which are the same for cognate and near-cognate codons. Increasing Mg2+ concentration decreases the accuracy by slowing dissociation of T-3 from its initial codon-and aminoglycoside-independent binding state on the ribosome. The distinct accuracy-corrupting mechanisms for aminoglycosides and Mg2+ ions prompted us to re-interpret previous biochemical experiments and functional implications of existing high resolution ribosome structures. We estimate the upper thermodynamic limit to the accuracy, the 'intrinsic selectivity' of the ribosome. We conclude that aminoglycosides do not alter the intrinsic selectivity but reduce the fraction of it that is expressed as the accuracy of initial selection. We suggest that induced fit increases the accuracy and speed of codon reading at unaltered intrinsic selectivity of the ribosome.

1 - 33 of 33
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