Ribosomens basala mekanismer

Tidsperiod: 2016-01-01 till 2018-12-31

Finansiär: Vetenskapsrådet

Bidragstyp: Projektbidrag

Budget: 2 300 000 SEK

We propose to study the structure-function relations of the bacterial ribosome. The ribosome is a large (2.5 MD) macromolecular complex that makes peptide chains with amino acid sequences as prescribed by the genes in the DNA of the genome. The genes are transcribed into messenger RNAs, which on the ribosome are red by amino acid carrying transfer RNAs. Major ribosomal cycles are initiation of messenger RNA translation, transfer RNA binding and peptide bond formation, translocation of messenger RNA and transfer RNA, termination of protein synthesis and ribosomal recycling back to a new round of initiation. One major purpose of the project will be to anchor kinetics data in a deeper understanding of the structural features of the ribosome that generate them, as briefly exemplified in the three following paragraphs.The accuracy of codon selection by tRNAs depends on one initial selection step and, as we discovered in the previous project period, two proofreading steps! We will extend present knowledge of ribosomal accuracy to a greatly expanded part of the genetic code and show how the accuracy is affected by tRNA modifications, antibiotic drugs and ribosomal mutations. We will also relate accuracy parameters to structural features of the mRNA translation machinery.In preliminary experiments we have recently demonstrated with Prof. J. Frank (Columbia U., N.Y.) that time resolved cryo-EM at high resolution can be used to monitor the authentic ribosomal complexes that are just about to be split into subunits. This type of experiment, which has become technically feasible only during the past year, has opened a new window to study complex structures at high resolution in their very functional acts. This will give unprecedented means to integrate structural and kinetics data in coherent theoretical concepts of the working principles of the ribosome. Supported by these preliminary data we will use traditional kinetics as well as time resolved cryo-EM to also address previously unanswered questions regarding the working principles of initiation of mRNA translation, mRNA translocation and termination of protein synthesis. We will also study how these ribosomal steps are affected by antibiotic drugs. Another major purpose of this proposal is to use our biochemical knowledge of ribosome function together with systems biological modelling to gain a deeper understanding of bacterial physiology and evolution. We propose to use systems biology modelling to predict the growth inhibitory action of so called slow inhibitors of ribosome function, like the antibiotics fusidic acid and viomycin. Such inhibitors dissociate very slowly from their ribosomal target, which leads to ribosomal queuing on messenger RNAs and adds a novel inhibitory function to these drugs. The proposed project deals with a macromolecular complex under extreme selection pressure for speed, size reduction and precise function; studying the ribosome provides keys to understanding the origin of present day life forms, the evolution of complex structures and to ribosome related human disease. Ribosomes are targets for 50% of all antibiotics in clinical use and in depth understanding of ribosome function will be essential for the creation of novel antibiotics and the slowing down of drug resistance among pathogens. Time plan – 2016: Genetic code translation (1 post-doc), Ribosomal recycling, including time resolved cryo-EM (1 post-doc). Proteome responses and slow inhibitors, theory and preliminary experiments (1 post-doc, ½ researcher). 2017: Genetic code translation: mutations, modifications, evolution (1 post-doc). Structure and kinetics of translocation and termination (1 post-doc), Proteome responses and slow inhibitors: evolutionary experiments and theory development (1 post doc, 1 Researcher). 2018: Genetic code translation: antibiotic drugs and evolution (1 post-doc, ½ researcher). Structure and kinetics of termination and initiation of protein s