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PMID: 9237903 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Expression of a coronavirus ribosomal frameshift signal in Escherichia coli: influence of tRNA anticodon modification on frameshifting.

Journal of molecular biology ·Vol. 270 ·No. 3 ·1997-07-18 ·Pages 360-73

Brierley I, Meredith MR, Bloys AJ, Hagervall TG

Abstract

Eukaryotic ribosomal frameshift signals generally contain two elements, a heptanucleotide slippery sequence (XXXYYYN) and an RNA secondary structure, often an RNA pseudoknot, located downstream. Frameshifting takes place at the slippery sequence by simultaneous slippage of two ribosome-bound tRNAs. All of the tRNAs that are predicted to decode frameshift sites in the ribosomal A-site (XXXYYYN) possess a hypermodified base in the anticodon-loop and it is conceivable that these modifications play a role in the frameshift process. To test this, we expressed slippery sequence variants of the coronavirus IBV frameshift signal in strains of Escherichia coli unable to modify fully either tRNA(Lys) or tRNA(Asn). At the slippery sequences UUUAAAC and UUUAAAU (underlined codon decoded by tRNA(Asn), anticodon 5' QUU 3'), frameshifting was very inefficient (2 to 3%) and in strains deficient in the biosynthesis of Q base, was increased (AAU) or decreased (AAC) only two-fold. In E. coli, therefore, hypomodification of tRNA(Asn) had little effect on frameshifting. The situation with the efficient slippery sequences UUUAAAA (15%) and UUUAAAG (40%) (underlined codon decoded by tRNA(Lys), anticodon 5' mnm5s2UUU 3') was more complex, since the wobble base of tRNA(Lys) is modified at two positions. Of four available mutants, only trmE (s2UUU) had a marked influence on frameshifting, increasing the efficiency of the process at the slippery sequence UUUAAAA. No effect on frameshifting was seen in trmC1 (cmnm5s2UUU) or trmC2 (nm5s2UUU) strains and only a very small reduction (at UUUAAAG) was observed in an asuE (mnm5UUU) strain. The slipperiness of tRNA(Lys), therefore, cannot be ascribed to a single modification site on the base. However, the data support a role for the amino group of the mnm5 substitution in shaping the anticodon structure. Whether these conclusions can be extended to eukaryotic translation systems is uncertain. Although E. coli ribosomes changed frame at the IBV signal (UUUAAAG) with an efficiency similar to that measured in reticulocyte lysates (40%), there were important qualitative differences. Frameshifting of prokaryotic ribosomes was pseudoknot-independent (although secondary structure dependent) and appeared to require slippage of only a single tRNA.

MeSH Terms
Anticodon/genetics Base Sequence Escherichia coli/genetics Frameshifting, Ribosomal/genetics Infectious bronchitis virus/genetics Molecular Sequence Data Nucleic Acid Conformation Plasmids/genetics Point Mutation RNA, Transfer, Asn/genetics RNA, Transfer, Lys/genetics RNA, Viral/chemistry,genetics
Chemicals
Anticodon RNA, Transfer, Asn RNA, Transfer, Lys RNA, Viral
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Brierley I
Department of Pathology, University of Cambridge, UK.
Meredith M R
Bloys A J
Hagervall T G
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Article Info
Journal
Journal of molecular biology
Abbr.
J Mol Biol
ISSN
0022-2836
DOI
10.1006/jmbi.1997.1134
Published
1997-07-18
Pages
360-73
Language
English
Region
England
NLM ID
2985088R
PMCID
PMC7126968
Subset
IM
External Links
PubMed source DOI: 10.1006/jmbi.1997.1134 PMC Full Text View journal details
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