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Studies on translation initiation and gene expression in Escherichia coli
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In prokaryotes, several mRNA sequences surrounding the initiation codon have been found to influence the translation process; these include the downstream region and its codon context, the Shine-Dalgarno sequence and the S1 ribosomal protein-binding site. In this thesis, the purpose has been to study the role of the downstream region and Shine-Dalgarno-like sequences on early translation elongation and gene expression in Escherichia coli.

The downstream region (DR) after the initiation codon (around five to seven codons), has an important role in the initiation of translation. We find that most of the codons which give very low gene expression at +2 (considering AUG as +1), reach 5 to 10 fold higher expression when those codons are positioned posteriori to +2, with the exception of the NGG codons. The NGG codons abort the translation process if located within the first five codons of the DR, due to peptidyl-tRNA drop-off. However, when the NGG codons are situated further down from the DR, the protein expression was increased at the same level of expression as in the presence of any other codon.

The Shine-Dalgarno (SD) is an important region of initiation in translation of bacteria. In spite of this, it has been found that Gram-negative bacteria could translate mRNAs with weak or non-functional SD, while the DR carries out a main role in the efficiency of translation. In addition, positions of SD and SD-like sequences are very important to direct initiation of translation in the choice between two possible initiation codons. A strong SD between two initiation sites will favor the second initiation site if it consists of a canonical start codon followed by a good DR.

The results suggest that the mRNA sequences surrounding the initiation codon: the downstream region and the Shine-Dalgarno and SD-like sequences, are very important contributors to the translation level and gene expression in Escherichia coli.

Place, publisher, year, edition, pages
Stockholm: Institutionen för genetik, mikrobiologi och toxikologi , 2006. , 50 p.
Keyword [en]
Downstream region, NGG codons, abortive translation, drop-off, Shine-Dalgarno sequence, gene expression and Escherichia coli
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:su:diva-985ISBN: 91-7155-214-6 (print)OAI: oai:DiVA.org:su-985DiVA: diva2:200784
Public defence
2006-05-10, Nordenskiöldsalen, Geovetenskapens hus U, Svante Arrhenius väg 8 B, Stockholm, 13:00
Opponent
Supervisors
Available from: 2006-04-19 Created: 2006-04-19Bibliographically approved
List of papers
1. A codon window in mRNA downstream of the initiation codon where NGG codons give strongly reduced gene expression in Escherichia coli
Open this publication in new window or tab >>A codon window in mRNA downstream of the initiation codon where NGG codons give strongly reduced gene expression in Escherichia coli
2004 In: Nucleic Acids Reseach, Vol. 32, no 17, 5198-5205 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-25906 (URN)
Note
Part of urn:nbn:se:su:diva-985Available from: 2006-04-19 Created: 2006-04-19Bibliographically approved
2. Abortive translation caused by peptidyl-tRNA drop-off at NGG codons in the early coding region of mRNA
Open this publication in new window or tab >>Abortive translation caused by peptidyl-tRNA drop-off at NGG codons in the early coding region of mRNA
2005 In: FEBS J, Vol. 272, no 20, 5306-5316 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-25907 (URN)
Note
Part of urn:nbn:se:su:diva-985Available from: 2006-04-19 Created: 2006-04-19Bibliographically approved
3. Influences on gene expression in vivo by a Shine-Dalgarno sequence
Open this publication in new window or tab >>Influences on gene expression in vivo by a Shine-Dalgarno sequence
Show others...
2006 (English)In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 60, no 2, 480-492 p.Article in journal (Refereed) Published
Abstract [en]

The Shine-Dalgarno (SD+: 5'-AAGGAGG-3') sequence anchors the mRNA by base pairing to the 16S rRNA in the small ribosomal subunit during translation initiation. We have here compared how an SD+ sequence influences gene expression, if located upstream or downstream of an initiation codon. The positive effect of an upstream SD+ is confirmed. A downstream SD+ gives decreased gene expression. This effect is also valid for appropriately modified natural Escherichia coli genes. If an SD+ is placed between two potential initiation codons, initiation takes place predominantly at the second start site. The first start site is activated if the distance between this site and the downstream SD+ is enlarged and/or if the second start site is weakened. Upstream initiation is eliminated if a stable stem-loop structure is placed between this SD+ and the upstream start site. The results suggest that the two start sites compete for ribosomes that bind to an SD+ located between them. A minor positive contribution to upstream initiation resulting from 3' to 5' ribosomal diffusion along the mRNA is suggested. Analysis of the E. coli K12 genome suggests that the SD+ or SD-like sequences are systematically avoided in the early coding region suggesting an evolutionary significance.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-25908 (URN)10.1111/j.1365-2958.2006.05110.x (DOI)000236328700020 ()
Available from: 2006-04-19 Created: 2006-04-19 Last updated: 2013-08-08Bibliographically approved

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