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Translational termination, frameshift and ribosome assembly
Stockholm University, Faculty of Science, Department of Microbiology.
1991 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The aim of this thesis was to study components involved in translational termination. Most of the work is centered around this, but the outcome of some of the experiments, led me onto other paths too.

Firstly, isolation of a mutant which was shown to be in release factor one, was mapped to min 26,5 on the Escherichia coli chromosome. The gene was named prfA and the mutant allele prfAl. The mutated RF1 is most probably impaired in its binding to the ribosome and not in its ability to recognize the termination site. This leads to a slower termination rate and allows a competing ternary complex to come in and read the stop codon. This can be studied as suppression and increased efficiency of UAG and UAA tRNA-suppressors.

By compiling all available E. coli sequences, some determinants for context have been inferred, with their main feature residing in the neighbouring codons, probably the 3' nucleotide. However, some element influencing the termination event, resides outside this immediate neighbourhood on the 5' side. Experiments performed in this thesis, suggest that the 3' determinant can be either U or C for the stop codon UAG, and that the 5' context effect is mediated through the peptidyl-tRNA in the P-site of the ribosome.

Secondly, it is known that ribosomal frameshift occurs at hot spots. As such the stop codon at the end of the frameshift window has been suggested. This hypothesis was tested on some frameshift mutations which are spontaneously suppressed and where the window ends with a UGA codon. If the compensating frameshift occurs at the stop codon it should be possible to decrease its suppression by addition of more release factor. It was shown that overproduction of active RF2 and not RF1 decreases the spontaneous suppression of the three frameshift mutations tested. This indicates that the stop codon can induce frameshift and thus, many experiments estimating frameshift frequency are likely to be overestimates.

Thirdly, a mutant isolated as a misreader, was shown to map in the gene for ribosomal protein S20, rpsT. The misreading characteristic is only part of a pleiotropic phenotype caused by truncated protein S20. Since S20 is one of the primary binding proteins in subunit assembly, the main defect is in formation of 30S subunits and their association with 50S subunits to form 70S ribosomes. It is also shown that two modifications, m62A and m5C, are essential for formation of active ribosomes in the mutant.

Place, publisher, year, edition, pages
Stockholm: Stockholm University, 1991. , p. 43
National Category
Microbiology
Identifiers
URN: urn:nbn:se:su:diva-165153Libris ID: 7608914ISBN: 91-7146-892-7 (print)OAI: oai:DiVA.org:su-165153DiVA, id: diva2:1281326
Public defence
1991-05-24, Hörsal G, Biologihus F, Svante Arrheniusväg 16, Stockholm, 10:00
Note

Härtill 5 uppsatser

Available from: 2019-01-22 Created: 2019-01-22 Last updated: 2019-01-22Bibliographically approved

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