Quality control of translation on the ribosome
Final Report Abstract
The major goal of the project was to understand how the ribosome selects its substrates for translation at the steps of initiation and elongation of protein synthesis. During initiation, the ribosome selects mRNAs and a reading frame on a given mRNA for translation, which affects the composition of the cellular proteome and defines the response to changing environmental conditions. We determined the order and timing of events during translation initiation in bacteria using a toolbox of fluorescence labeled components and monitoring changes in fluorescence and FRET. We identified several steps which can serve to monitor and select a particular mRNA for translation based on the features of its 5’ UTR. The efficiency of mRNA selection, i.e. how often it will be selected for translation, depends on a kinetic partitioning between forward reactions that proceed towards the productive 70S initiaton complex versus dissociation of the mRNA or a temporary stalling at an early, yet unproductive step of initiation. Different features of the mRNA, such as the properties of the Shine-Dalgarno sequence, secondary structure elements at the ribosomes binding site, or the presence of the initiator codon, are sensed at different stages, resulting in a multidimensional selection landscape that can be regulated by intra- and extracellular factors. The established toolbox will allow us in the future to examine the mechanism of translational initiation on mRNAs that have no Shine-Dalgarno sequence or no 5’ UTR at all, and to investigate loading of ribosomes in a polysome, initiation on overlapping genes within an operon, regulation of riboswitches that are operated at the translation level, or coupling between transcription and translation. We obtained the cryo-EM structure of the 30S initiation complex with all three initiation factors present. Further work would be necessary to improve the resolution of the cryo-EM and to analyze the dynamics of the ribosome during initiation. In the second part of the project we asked the question of how the ribosomes maintain high fidelity of tRNA selection, while at the same time ensuring rapid translation. Our data suggest that the GTP hydrolysis step is crucial for the optimization of both the speed and accuracy, which explains the necessity for the trade-off between the two fundamental parameters of translation. The values of elemental rate constants, which we now provide for two sets of experimental conditions, will be used in mathematical modeling of translation in vitro and in vivo. Finally, we started the project aimed at understanding recoding events during which the ribosome overcomes the universal rules of standard decoding. As examples, we chose programmed –1 ribosomal frameshiftingon the Infectious Bronchitis Virus model mRNA and bypassing of 50 nucleotides during translation of gene 60 mRNA of bacteriophage T4. The results imply that during recoding the dynamics of the ribosome – as manifested by kinetics of elemental reactions – is altered by the mRNA elements (secondary structure elements, specific mRNA sequences), indicating that the mRNA carries the secondary layer of information for the recoding events. These findings provide insights into the function and evolution of the ribosomes. Future progress in understanding the kinetics of recoding will show whether the ribosome’s capacity for alternative interpretation of mRNA can be utilized for engineering controlled expression of designer proteins.
Publications
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(2010) Optimization of speed and accuracy of decoding in translation. EMBO J. 29, 3701-3709
Wohlgemuth, I., Pohl, C., and Rodnina, M.V.
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(2010) Ribosome-bound initiation factor IF2 recruits initiator tRNA to the 30S initiation complex. EMBO Rep. 11, 312-316
Milon, P., Carotti, M., Konevega, A.L., Wintermeyer, W., Rodnina, M.V., Gualerzi, C.O.
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(2011) Distortion of tRNA upon near-cognate codon recognition on the ribosome. J. Biol. Chem. 286, 8158-8164
Mittelstaet, J., Konevega, A.L., and Rodnina M.V.
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(2011) The cryo-EM structure of a complete 30S translation initiation complex from Escherichia coli. PLoS Biology 9, e1001095
Julián, P., Milon, P., Agirrezabala, X., Lasso G., Gil D, Rodnina, M.V., and Valle, M.
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(2012) Impact of methylations of m2G966/m5C967 in 16S rRNA on bacterial fitness and translation initiation. Nucl. Acids Res. 40, 7885-7895
Burakovsky D.E., Prokhorova I.V., Sergiev P.V., Milón P., Sergeeva O.V., Bogdanov A.A., Rodnina M.V., Dontsova O.A.
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(2012) Real-time assembly landscape of bacterial 30S translation initiation complex. Nat. Struct. Mol. Biol. 19, 609-615
Milón, P., Maracci C., Filonava L., Gualerzi C.O., Rodnina M.V.
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(2013) tRNA tKUUU, tQUUG, and tEUUC wobble position modifications fine-tune protein translation by promoting ribosome A-site binding. Proc. Natl. Acad. Sci. USA 110, 12289-94
Rezgui, V., Tyagi, K., Ranjan, N., Konevega, A.L., Mittelstaet, J., Rodnina, M.V., Peter, M., Pedroli, P.G.
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(2014) High-efficiency translational bypassing of non-coding nucleotides specified by mRNA structure and nascent peptide. Nat. Communs., 5, Article number: 4459 (2014)
Samatova, E., Konevega, A. L., Wills, N. M., Atkins, J. F., Rodnina, M. V.
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(2014) Programmed –1frameshifting by kinetic partitioning during impeded translocation. Cell157, 1619-1631
Caliskan, N., Katunin, V. I., Belardinelli, R., Peske, F., Rodnina, M. V.
