EXPRESSION OF A CORONAVIRUS RIBOSOMAL FRAMESHIFT SIGNAL IN ESCHERICHIA-COLI - INFLUENCE OF TRANSFER-RNA ANTICODON MODIFICATION ON FRAMESHIFTING

Eukaryotic ribosomal frameshift signals generally contain two elements , a heptanucleotide slippery sequence (XXXY<(YYN)under bar>) and an RN A secondary structure, often an RNA pseudoknot, located downstream. Fr ameshifting takes place at the slippery sequence by simultaneous slipp age of two ribosome-bound tRNAs. All of the tRNAs that are predicted t o decode frameshift sites in the ribosomal A-site (XXXY<(YYN)under bar >) possess a hypermodified base in the anticodon-loop and it is concei vable that these modifications play a role in the frameshift process. To test this, we expressed slippery sequence variants of the coronavir us IBV frameshift signal in strains of Escherichia coli unable to modi fy fully either tRNA(Lys) or tRNA(Asn). At the slippery sequences UUUA <(AAC)under bar> and UUUA<(AAU)under bar> (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 i ncreased (AAU) or decreased (AAC) only two-fold. In E, coli, therefore , hypomodification of tRNA(Asn) had little effect on frameshifting. Th e situation with the efficient slippery sequences UUUA<(AAA)under bar> (15%) and UUUA<(AAG)under bar> (40%) (underlined codon decoded by tRN A(Lys), anticodon 5' mnm(5)s(2)UUU 3') was more complex, since the wob ble base of tRNA(Lys) is modified at two positions. Of four available mutants, only trmE (s(2)UUU) had a marked influence on frameshifting, increasing the efficiency of the process at the slippery sequence UUUA <(AAA)under bar>. No effect on frameshifting was seen in trmC1 (cmnm(5 )s(2)UUU) or trmC2 (nm(5)s(2)UUU) strains and only a very small reduct ion (at UUUA<(AAG)under bar> was observed in an asuE (mnm(5)UUU) strai n. The slipperiness of tRNA(Lys), therefore, cannot be ascribed to a s ingle modification site on the base. However, the data support a role for the amino group of the mnm(5) substitution in shaping the anticodo n structure. Whether these conclusions can be extended to eukaryotic t ranslation systems is uncertain. Although E. coli ribosomes changed fr ame at the IBV signal (UUUAAAG) with an efficiency similar to that mea sured in reticulocyte lysates (40%), there were important qualitative differences. Frameshifting of prokaryotic ribosomes was pseudoknot-ind ependent (although secondary structure dependent) and appeared to requ ire slippage of only a single tRNA. (C) 1997 Academic Press Limited.