Table of contents:
The role of the ribosomal stalk protein L7/L12 in the interaction with translation factors
Chandra Sekhara Mandava, Ph.D.
Xueliang Ge, Ph.D.
Ranjeet Kumar, Chenhui Huang, Josefine Ederth, Former Postdocs
Christiane Lupczyk, Former Project Student
In bacteria, the ribosomal 'stalk' consists of two L7/L12 protein dimers and one L10 protein attached to the rRNA next to the protein L11. The L7/L12 proteins are highly flexible and change conformation in different steps of translation. These proteins interact with the translational G-factors such as EF-G and EF-Tu, and may be involved in GTP binding and in activation of factor dependent GTP hydrolysis. However, the molecular mechanism of these processes is quite unclear. The specific goal of this project is to characterize the role of L7/L12 in interactions with translational G-proteins. Using heteronuclear NMR, we have mapped the residues on L7/L12 that are involved directly in interaction with IF2, EF-G, EF-Tu and RF3. We will also map the regions on the factors involved in these interactions as the next step. Another aim is to study whether L7/L12 acts as GTPase activator for IF2 and RF3 as it does for EF-G and EF-Tu. We are also studying the effect of depletion of ribosomal stalk proteins (L7/L12, L8 and L11) in IF2 dependent subunit association.We have produced an E. coli strain, which has ribosomes with only one L7/L12 dimer. In vivo and in vitro characterization of these ribosomes are ongoing.
The fusidic acid resistance in Staphylococcus aureus.
Ravi Kiran Koripella, Ph.D. Student
Mikael Holm, Ph.D Student
Hasanthi Karunasekera, Jenny Honek Former Project Students
Chenhui Huang, Musturi Venkataramana, Former Postdocs
Key investigator: Suparna Sanyal
Fusidic acid is an antibiotic, clinically used to prevent Staphylococcus aureus infection since last four decades. It binds to EF-G and blocks EF-G.GDP on the ribosome in the post-translocation state. So far all fusidic acid related studies are done with E. coli and some other thermophilic bacteria, which are not the natural targets for fusidic acid and it is practically impermeable in these hosts. This is the first concerted effort to study the fusidic acid inhibition and resistance mechanism in vitro using the EF-Gs from Staphylococcus aureus, the actual drug target of fusidic acid. Our goals are a)To locate the fusidic acid binding site on EF-G. b)To characterize the fusidic acid resistant mutants of Staphylococcus aureus, particularly those which have gained biological fitness by additional mutations in the fusA gene.
Ribosome assisted protein folding and it's implication in prion diseases
Yanhong Pang, Ph.D. Student
Petar Kovachev, Ph.D. Student
Debapriya Banerjee, Ph.D.
Venkata Sriram Kurella Ariane Schabe, Neelanjan Vishnu Former Project students
Suzana DosReis, Former Postdoc
Debasis Das, Erik Richter, Helena Kristiansson, Hiroki Kawahara Former Project /Visiting Students
Collaboration with Marc Blondel Group, Brest, France
Recently our understanding of interplay between structure and function of bacterial ribosome has increased significantly. One major question that has remained unanswered, is how newly synthesized polypeptide chains are folded in the living cells. Several molecular chaperones have been shown to be part of the process. But it has also been demonstrated in vitro that ribosomes from archaebacteria, eubacteria, animals, plants and mitochondria can refold a large number of denatured proteins to their active state all the different proteins which have been subjected to ribosomal refolding show remarkable activity recovery compared to spontaneous folding (Das B et al, 1996). The ribosome assisted protein folding activity have been assigned to the large subunit 50S, and more precisely to the domain V of the 23SrRNA. The aim of the project is to try to understand how this folding occurs, is it co or posttranslational? Does this folding happen on the translating ribosome or in trans? Is there any universal binding motif on peptides that are recognized by the ribosome? Is there any implication of this function in prion-diseases?
The prions are infectious proteins that cause a group of fatal neurodegenerative disease like Creutzfeld-Jacob or BSE (Mad Cow Disease). These diseases involve the conversion of the normal form of the protein (PrPc) into the disease causing isoform (PrPsc) and are recognized as a 'protein-folding associated problem'. Recently we have shown in collaboration with Marc Blondel group in France, that two antiprion drugs 6AP and GA use ribosomes as the molecular target in the cell (Tribouillard- Tanvier D et al, 2008). These drugs bind specifically to ribosomal RNA from the large subunit and inhibit the folding activity of the ribosome, but not protein synthesis. The detailed characterization of the process is ongoing.
In a recent in vitro study it has been shown that the unfolded proteins can dissociate 70S ribosome into the subunits while refolding. In another study, the nascent protein chain is seen to be associated with the 50S subunit immediately after its synthesis. This implicates that the nascent protein chain may have a role in translation termination and the dissociation of the ribosomal subunits. At present we are studying the release and folding of a full-length protein chain using a cell-free protein synthesis system.
Translation termination in bacteria
Xueliang Ge, Ph.D
Gürkan Korkmaz, Ph.D Student
Ravi Kiran Koripella, Ph.D
Chenhui Huang, Former Postdoc
Nhan Tran Van, Anders Bankefors, Chen Chen Former Project Student
Collaboration with Joachim Frank Group, New York, USA and Valerie Heurgue-Hamard, CNRS Paris, France
In the termination process of translation the newly synthesized protein is being released from the ribosome. In this step of translation two class I release factor (RF) overlap partly in their codon recognition and are being recycled by a class II release factor namely RF3.
We are approaching the mechanism of translation termination by investigating the action of RF3 involving possible interaction with RF1/2 and ribosomal protein L12. Further we are interested in the decoding of stop codons by class I release factors. Recently a computational approach was used to simulate and calculate the binding energy of several amino acids of RF1/2, in this project we want to compare these results with our experimental mutagenesis. Additionally we published a database analyzing the stop codon usage in bacteria.
Translation in Mycobacteria
Xueliang Ge Ph.D
Emelie Gabrielsson Former Master Student
Ranjeet Kumar Former Postdoc
Translocation and RNA
Mikael Holm, Ph.D Student
Reconstituted Transcription Translation Folding System
Kristin Peisker, Ph.D
Sunanda Chatterjee, PhD
Jan Pille, Project Student, Antje Wanglin former M. Sc. Research Assistant
Reconstituted Transcription Translation folding (RTTF) system for cell free protein synthesis:
We have been successful to reconstitute a cell-free transcription-translation-folding system (named as RTTF system) using purified translation components from bacteria E. coli, where active proteins are being synthesized with good efficiency. So far, we have produced firefly-luciferase, dihydrofolate reductase (DHFR), fluorescent proteins mCherry and turbo-GFP in the system starting from DNA or in vitro transcribed mRNA. The system provides great flexibility to test individual translational components with mutations or with specific modulators. Some examples are ribosomes with altered stalk protein complex (Mandava et al., NAR, 2012); L1 deleted ribosome (Tobin et al., under preparation) and fusidic acid resistant mutant EF-Gs from pathogenic bacteria Staphylococcus aureus. Further, the system can be used for checking accuracy in protein synthesis, frame-shifting defects as well as for studying refolding and maturation of the target proteins. Currently, a similar system with Mycobacterial translation machinery is under construction, which will be used for high-throughput screening of potential TB drugs. Last but not the least, this system provides ample scope to produce unique peptides and engineered proteins which can be of great value for biomedical and technical applications.
The system has been highly evaluated by the international expert panel appointed by The Swedish Research Council and Formas. Read the comment quoted from the Draft Midterm Evaluation Report of the 2006 Linnaeus environments and Doctoral programs for UPPSALA RNA RESEARCH CENTER
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