Functional studies of E. coli ribosomes and characterization of mini open reading frames
2000 (English)Doctoral thesis, comprehensive summary (Other academic)
In this thesis, problems concerning the elongation, termination and recycling steps of prokaryotic translation have been addressed.
An explanation is given for growth inhibition due to minigene expression.Minigene transcripts containing very short open reading frames inhibit proteinsynthesis in E. coli strains partially defective in peptidyl-tRNA hydrolase (Pth) activity. Several parameters contribute to minigene toxicity: i) the dissociation rate of short peptidyl-tRNAs from the ribosome is very high ii) release factor dependent rate of hydrolysis of the short peptidyl-tRNAs on the ribosome is very slow iii) the hydrolysis of short peptidyl-tRNAs by Pth(rap) mutant is inefficient. A linear correlation between a toxicity index, ITox, defined from these biochemical measurements and a toxicity parameter calculated from growth inhibition is obtained.
Minigenes with efficient Shine/Dalgamo sequences are detrimental to Pthwild-type cells of E. coli. Short mRNA transcripts remain associated with theribosome after each termination event during many translation cycles. The probability that a ribosome reinitiates on the same messenger RNA molecule decreases with the length of the open reading frame.
Overproduced E. coli polypeptide release factor 2 (RF2) is impaired intranslation termination. A post-translational modification on a glutamine residue was identified in chromosomally expressed RF2 but not in the overproduced protein. This modification increases the RF2 dependent catalytic rate (kcat) of hydrolysis of the peptidyl-tRNA on the ribosome more than ten fold. A difference in the amino acid sequence of chromosomally expressed RF2 proteins from two strains of E. coli was identified. Substitution of an alanine residue to a threonine in RF2 lowers the stop codon dependent rate of hydrolysis of short peptidyl-tRNAs on the ribosome considerably.
Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2000. , 44 p.
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 524
Cell and molecular biology
Cell- och molekylärbiologi
Biochemistry and Molecular Biology
Research subject Molecular Biology
IdentifiersURN: urn:nbn:se:uu:diva-1046ISBN: 91-554-4692-2OAI: oai:DiVA.org:uu-1046DiVA: diva2:160584
2000-05-11, Lecture room B42, Biomedical Center (BMC), Uppsala, Uppsala, 14:00