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

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.

Transfer RNA modifications that alter +1 frameshifting in general fail to affect −1 frameshifting

“…According to the simultaneous slippage model (Jacks et al 1988), −1 frameshifts occur after the A-site is filled, by simultaneous slippage of the tRNAs present in both A-and P-sites, that is prior to translocation. In general, our results (Table 3) and those of others (Hagervall et al 1993;Brierley et al 1997;Carlson et al 1999;Marczinke et al 2000) sug- gest that tRNA modifications have no or only a minor effect on −1 frameshifts. In fact, in some cases, the presence of the modified nucleoside actually stimulates −1 frameshifting (Fig.…”

“…Similarly, in E. coli, the s 2 -or the mnm 5 -group each has very little or no influence on −1 frameshifting at a U-UUA-AAG site, and the s 2 -group has no influence on −1 frameshifting at the U-UUA-AAA site (Brierley et al 1997). However, lack of the mnm 5 -group increases the −1 frameshifting at the slippery U-UUA-AAA sites twofold (Brierley et al 1997). Thus, contrary to the large impact of mnm 5 s 2 U34 to prevent +1 shifts, its role in −1 frameshifting is minor, and, in fact, in some cases the presence of it increases the −1 frameshift error.…”

“…Using the same in vitro system, the level of −1 frameshifting at the A-AAA-AAC site only increased 1.5-fold when the Q-deficient tRNA Asn GUU from yeast was added to the lysate (Carlson et al 2001). When the effect by Q34 was analyzed in vivo using a Q34 deficient mutant of E. coli, frameshifting by tRNA Leu U?AA decreased 2-fold at the U-UUA-AAC site (i.e., when tRNA Leu U?AA interacted with the AAC codon) and increased 2-fold at the U-UUA-AAU site (i.e., when tRNA Leu U?AA interacted with the AAU codon; Brierley et al 1997). The difference between those and our ( Fig.…”

Translational Control of Viral Gene Expression in Eukaryotes

“…The process of frameshifting was first described from studies of Rous sarcoma virus replication (220) and has been extensively defined in the MMTV and infectious bronchitis virus (IBV) (a coronavirus)] systems, although it occurs widely throughout other eukaryotic RNA viruses (151). Frameshift sites within the viral mRNA correspond to heptanucleotide sequences in which the mRNA slips 1 base with respect to the tRNAs in the A and P sites on the translating ribosome (40,108). The frameshift site, also known as the slippery site, allows the tRNA to move along the mRNA template by 1 base (forward or back) and reestablish codon-anticodon pairing, resulting in a stable ϩ1 or Ϫ1 reading frame shift.…”

“…Thus, it has been suggested that hypomodified variants of these tRNAs may function to promote shifting by being less bulky and therefore more easily moved within the slippery site (186). However, this idea remains controversial, since other researchers have proposed that frameshifting is mediated by standard cellular tRNAs and is simply dependent on the strength of the codon-anticodon tRNA interaction (40,465). In either case, frameshifting requires a pseudoknot structure near the slippery site to stimulate the frameshifting events.…”