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Ribosomal Stalk Protein L12: Structure, Function and Application
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. (Dr. Suparna Sanyal)
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Ribosomal stalk proteins are known to play important role in protein synthesis. The ‘stalk’, an extended structure on the large subunit of the ribosome is composed mainly of two to three dimers of L12 and one L10 protein, which forms the base of the stalk. In E. coli, four copies of L12 molecules exist as dimer of dimers forming the pentameric L8 complex together with L10. This thesis is a collection of four interlinked studies on the structure, function and application of the ribosomal stalk protein L12. In the first study, we have mapped the interaction sites of the four major translation GTPase factors (IF2, EF-Tu, EF-G & RF3) on L12 molecule using heteronuclear NMR spectroscopy. Surprisingly, all these factors produced an overlapping interaction map spanning two α-helices on the C terminal domain of L12, thereby suggesting a general nature of the interaction between L12 and the GTPase factors. L12 is known to stimulate GTPase activity of the elongation factors EF-Tu and EF-G. Here, we have clarified the role of L12 in IF2 mediated initiation of protein synthesis. Our data suggest that rapid subunit association requires a specific interaction between the L12 protein on the 50S and IF2·GTP on the 30S preinitiation complex. We have also shown that L12 is not a GAP for IF2 and GTP hydrolysis triggers IF2 release from the 70S initiation complex. The next question we have addressed is why multiple copies of L12 dimer are needed on the ribosome. For this purpose, we created a pure E. coli strain JE105, where the terminal part of rplJ gene coding for the binding site of one L12 dimer on protein L10 was deleted in the chromosomal locus. Using ribosomes with single L12 dimer we have observed that the rate of the initiation and elongation involving IF2 and EF-G gets most compromised, which in turn decreases the growth rate of the bacteria.  This study also indicates that L12 can interact with different GTPase factors in a specialized manner. Lastly, we have developed an application making advantage of the multiple L12 dimers on the ribosome. By inserting a (His)6-tag at the C-terminus of the L12 protein we have created a novel E. coli strain (JE28), where all ribosomes are tetra-(His)6-tagged. Further, we have developed a single step method for purification of the active (His)6-tagged ribosomes from JE28.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2011. , 49 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 848
Keyword [en]
Ribosome, protein synthesis, L12 dimer, initiation, elongation, G factors, and His-tag
National Category
Biochemistry and Molecular Biology
Research subject
Biology with specialization in Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-157198ISBN: 978-91-554-8144-5 (print)OAI: oai:DiVA.org:uu-157198DiVA: diva2:438522
Public defence
2011-10-06, B41, BMC, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2011-09-15 Created: 2011-08-19 Last updated: 2011-11-03Bibliographically approved
List of papers
1. The ribosomal stalk binds to translation factors IF2, EF-Tu, EF-G and RF3 via a conserved region of the L12 C-terminal domain
Open this publication in new window or tab >>The ribosomal stalk binds to translation factors IF2, EF-Tu, EF-G and RF3 via a conserved region of the L12 C-terminal domain
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2007 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 365, no 2, 468-479 p.Article in journal (Refereed) Published
Abstract [en]

Efficient protein synthesis in bacteria requires initiation factor 2 (IF2), elongation factors Tu (EF-Tu) and G (EF-G), and release factor 3 (RF3), each of which catalyzes a major step of translation in a GTP-dependent fashion. Previous reports have suggested that recruitment of factors to the ribosome and subsequent GTP hydrolysis involve the dimeric protein L12, which forms a flexible "stalk" on the ribosome. Using heteronuclear NMR spectroscopy we demonstrate that L12 binds directly to the factors IF2, EF-Tu, EF-G, and RF3 from Escherichia coli, and map the region of L12 involved in these interactions. Factor-dependent chemical shift changes show that all four factors bind to the same region of the C-terminal domain of L12. This region includes three strictly conserved residues, K70, L80, and E82, and a set of highly conserved residues, including V66, A67, V68 and G79. Upon factor binding, all NMR signals from the C-terminal domain become broadened beyond detection, while those from the N-terminal domain are virtually unaffected, implying that the C-terminal domain binds to the factor, while the N-terminal domain dimer retains its rotational freedom mediated by the flexible hinge between the two domains. Factor-dependent variations in linewidths further reveal that L12 binds to each factor with a dissociation constant in the millimolar range in solution. These results indicate that the L12-factor complexes will be highly populated on the ribosome, because of the high local concentration of ribosome-bound factor with respect to L12.

Keyword
L12, ribosome, GTPase, NMR spectroscopy, protein synthesis
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-146545 (URN)10.1016/j.jmb.2006.10.025 (DOI)000243243200015 ()17070545 (PubMedID)
Available from: 2011-02-17 Created: 2011-02-17 Last updated: 2017-12-11Bibliographically approved
2. The Ribosomal Stalk Plays a Key Role in IF2-Mediated Association of the Ribosomal Subunits
Open this publication in new window or tab >>The Ribosomal Stalk Plays a Key Role in IF2-Mediated Association of the Ribosomal Subunits
2010 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 399, no 1, 145-153 p.Article in journal (Refereed) Published
Abstract [en]

Ribosomal "stalk" protein L12 is known to activate translational GTPases EF-G and EF-Tu, but not much is known about its role in relation to other two translational G factors, IF2 and RF3. Here, we have clarified the role of L12 in IF2-mediated initiation of bacterial protein synthesis. With fast kinetics measurements, we have compared L12-depleted 50S subunits with the native ones in subunit association, GTP hydrolysis, Pi (inorganic phosphate) release and IF2 release assays. L12 depletion from 50S subunit slows the subunit association step significantly (similar to 40 fold) only when IF2.GTP is present on the 30S preinitiation complex. This demonstrates that rapid subunit association depends on a specific interaction between the L12 stalk on the 50S subunit and IF2.GTP on the 30S subunit. L12 depletion, however, did not affect the individual rates of the subsequent steps including GTP hydrolysis on IF2 and Pi release. Thus, L12 is not a GTPase activating protein (GAP) for IF2 unlike as suggested for EF-G and EF-Tu.

Keyword
ribosome, translation initiation, L12, IF2, subunit association
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-136263 (URN)10.1016/j.jmb.2010.04.009 (DOI)000278779900012 ()
Available from: 2010-12-11 Created: 2010-12-11 Last updated: 2017-12-11Bibliographically approved
3. Bacterial ribosome requires multiple L12 dimers for efficient initiation and elongation of protein synthesis involving IF2 and EF-G
Open this publication in new window or tab >>Bacterial ribosome requires multiple L12 dimers for efficient initiation and elongation of protein synthesis involving IF2 and EF-G
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2012 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 40, no 5, 2054-2064 p.Article in journal (Refereed) Published
Abstract [en]

The ribosomal stalk in bacteria is composed of four or six copies of L12 proteins arranged in dimers that bind to the adjacent sites on protein L10, spanning 10 amino acids each from the L10 C-terminus. To study why multiple L12 dimers are required on the ribosome, we created a chromosomally engineered Escherichia coli strain, JE105, in which the peripheral L12 dimer binding site was deleted. Thus JE105 harbors ribosomes with only a single L12 dimer. Compared to MG1655, the parental strain with two L12 dimers, JE105 showed significant growth defect suggesting suboptimal function of the ribosomes with one L12 dimer. When tested in a cell-free reconstituted transcription-translation assay the synthesis of a full-length protein, firefly luciferase, was notably slower with JE105 70S ribosomes and 50S subunits. Further, in vitro analysis by fast kinetics revealed that single L12 dimer ribosomes from JE105 are defective in two major steps of translation, namely initiation and elongation involving translational GTPases IF2 and EF-G. Varying number of L12 dimers on the ribosome can be a mechanism in bacteria for modulating the rate of translation in response to growth condition.

Keyword
ribosome, translation initiation, L12, IF2, subunit association, protein synthesis
National Category
Biochemistry and Molecular Biology
Research subject
Biology with specialization in Molecular Biology
Identifiers
urn:nbn:se:uu:diva-157694 (URN)10.1093/nar/gkr1031 (DOI)000302019900022 ()22102582 (PubMedID)
Available from: 2012-03-13 Created: 2011-08-22 Last updated: 2017-12-08Bibliographically approved
4. A single-step method for purification of active His-tagged ribosomes from a genetically engineered Escherichia coli
Open this publication in new window or tab >>A single-step method for purification of active His-tagged ribosomes from a genetically engineered Escherichia coli
2009 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 37, no 2, e15- p.Article in journal (Refereed) Published
Abstract [en]

With the rapid development of the ribosome field in recent years a quick, simple and high-throughput method for purification of the bacterial ribosome is in demand. We have designed a new strain of Escherichia coli (JE28) by an in-frame fusion of a nucleotide sequence encoding a hexa-histidine affinity tag at the 3-end of the single copy rplL gene (encoding the ribosomal protein L12) at the chromosomal site of the wild-type strain MG1655. As a result, JE28 produces a homogeneous population of ribosomes (His)(6)-tagged at the C-termini of all four L12 proteins. Furthermore, we have developed a single-step, high-throughput method for purification of tetra-(His)(6)-tagged 70S ribosomes from this strain using affinity chromatography. These ribosomes, when compared with the conventionally purified ones in sucrose gradient centrifugation, 2D-gel, dipeptide formation and a full-length protein synthesis assay showed higher yield and activity. We further describe how this method can be adapted for purification of ribosomal subunits and mutant ribosomes. These methodologies could, in principle, also be used to purify any functional multimeric complex from the bacterial cell.

National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-137336 (URN)10.1093/nar/gkn992 (DOI)000262963400006 ()
Available from: 2010-12-15 Created: 2010-12-15 Last updated: 2017-12-11Bibliographically approved

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