Several viruses use an ef®cient À1 ribosomal frameshifting mechanism to control expression of their replicases. Frameshifts of this class were ®rst described as the process by which the gagpol polyprotein of the retrovirus Rous sarcoma virus (RSV) is expressed from the overlapping gag and pol open reading frames (ORFs: Jacks & Varmus, 1985) . Related frameshift signals have since been documented in an increasing number of systems, including several other retroviruses, a number of eukaryotic positive-strand RNA viruses, a double-stranded RNA virus of yeast, some plant RNA viruses and certain bacteriophage (reviewed by Brierley, 1995) . The phenomenon is not restricted to viruses; frameshift signals of the``retrovirus type'' have been described in a number of Escherichia coli insertion elements (reviewed by Chandler & Fayet, 1993; Farabaugh, 1996) and in a conventional cellular gene, the dna X gene of E. coli (Blinkowa & Walker, 1990; Flower & McHenry, 1990; Tsuchihashi & Kornberg, 1990; Tsuchihashi, 1991) . The mRNA signals that specify frameshifting appear to be composed of two essential elements; a heptanucleotide``slippery'' sequence, where the ribosome changes reading frame, and a region of RNA secondary structure, often in the form of an RNA pseudoknot, located a few nucleotides downstream (Jacks et al., 1988a; Brierley et al., 1989; ten Dam et al., 1990) . The molecular mechanism of the frameshift process is only poorly understood, but work from several groups supports a model (Jacks et al., 1988a) in which the elongating ribosome pauses upon encountering the region of mRNA secondary structure, facilitating realignment of the slippery sequence-decoding tRNAs in the À1 frame. The heptanucleotide stretch that forms the slippery sequence contains two homopolymeric triplets and conforms, in the vast majority of cases, to the motif XXXYYYN. Frameshifting at this sequence is thought to occur by simultaneous slippage of two ribosome-bound tRNAs, presumably peptidyl and aminoacyl tRNAs, which are translocated from the zero (X XXY YYN) to the À1 phase (XXX YYY: Jacks et al., 1988a) . Following the slip, the tRNAs remain base-paired to the mRNA in at least two out of three anticodon positions. There is considerable experimental support for this model, particularly from site-directed mutagenesis studies (Jacks et al., 1988a; Dinman et al., 1991; Dinman & Wickner, 1992; Brierley et al., 1992) , sequencing of trans-frame proteins (Hizi et al., 1987; Jacks et al., 1988a,b; Weiss et al., 1989; Nam et al., 1993) and nucleotide sequence comparisons (Jacks et al., 1988a; ten Dam et al., 1990) . The protein sequencing studies indicate that the frameshift occurs at the second codon of the tandem slippery pair, i.e. at that codon decoded in the ribosomal aminoacyl (A) site (XXXYYYN). The importance of the A-site tRNA in frameshift-ing was also apparent from the mutagenesis studies; point mutations affecting the A-site tRNA were generally more inhibitory than those affecting the P-site tRNA.
A key question that has remained unanswered is whether frameshifting at the slippery sequence is mediated by canonical tRNAs, or requires the participation of special``shifty'' tRNAs, more prone to frameshift than their``normal'' counterparts (Jacks et al., 1988a) . At naturally occurring frameshift sites, of the codons that are decoded in the ribosomal A-site prior to tRNA slippage (XXXYYYN), only ®ve are represented in eukaryotes, AAC, AAU, UUA, UUC and UUU, and two in prokaryotes, AAA and AAG (Farabaugh, 1996) . These codons are decoded by tRNAs with a highly modi®ed base in the anticodon loop (see Hat®eld et al., 1992 and references therein) . In tRNA Asn (AAC, AAU), the wobble base is queuosine (Q), in tRNA Phe (UUC, UUU), wyebutoxine (Y) is present just 3 H of the anticodon, in tRNA Lys (AAA, AAG), the wobble base is 5-methylaminomethyl-2-thiouridine (mnm 5 s 2 U) (prokaryotes) and in tRNA Leu (UUA), 2-methyl-5-formylcytidine is present at the wobble position (Debarros et al., 1996) . Hat®eld et al. (1992) have suggested that hypomodi®ed variants of these tRNAs may exist that function as speci®c``shifty'' tRNAs, since such variants will have a considerably less bulky anticodon and be more free to move around at the decoding site. Indirect support for this hypothesis comes from an examination of the modi®cation status of the anticodons of the aminoacyl-tRNAs that are required for translation at and around the frameshift sites of human immunode®ciency virus type 1 (HIV-1), human T-cell leukaemia virus type I (HTLV-1) and bovine leukaemia virus (BLV: Hat®eld et al., 1989) . It was found that in HIV-1 infected cells, most of the tRNA Phe lacked Y and in HTLV-1 and BLV infected cells, most of the tRNA Asn lacked Q. However, research from other groups has suggested an alternative hypothesis, in which frameshifting is mediated by standard cellular tRNAs (Tsuchihashi, 1991; Tsuchihashi & Brown, 1992; Brierley et al., 1992) . These authors propose that frameshifting at a particular site depends, amongst other parameters, upon the strength of the interaction between the slippery sequence codons and the tRNAs decoding it and that if this interaction is relatively weak, then slippage is more likely to occur. The strength of the interaction between mRNA and tRNA is likely to be in¯uenced considerably by the kind of base-pair that forms between the 3 H base of the codon and the 5 H base of the anticodon (position 34) at the wobble position. Modi®cation of the anticodon wobble base of frameshift site-decoding tRNAs may well in¯uence this interaction and hence the level of frameshifting observed. A prediction of the hypothesis is that the anticodon modi®cations present in tRNAs that decode highly ef®cient slippery sequences reduce recognition of the corresponding codons.
Here, we have attempted to test these hypotheses by measuring directly the in¯uence of tRNA anticodon modi®cation on frameshifting. Our approach was to determine the ef®ciency of the frameshift signal of the coronavirus infectious bronchitis virus (IBV; Brierley et al., 1987 Brierley et al., , 1989 in mutant strains of Escherichia coli unable to modify fully either tRNA Lys or tRNA Asn . The IBV frameshift signal, which is present at the overlap of the 1a and 1b ORFs of the virus genomic RNA, is a well-characterised eukaryotic system (Brierley et al., 1991 (Brierley et al., , 1992 ) that comprises the slippery sequence UUUAAAC and a downstream RNA pseudoknot. We began by determining the components of the IBV signal required for ef®cient frameshifting in E. coli and then proceeded with the investigation of the role of tRNA anticodon modi®cation in frameshifting. Four slippery sequence variants of the IBV site (UUUAAAX, where X was A, C, G or U) were tested, focusing speci®cally on the A-site decoding tRNAs. The results obtained indicate that in E. coli there is little in¯uence of the tRNA modi®cation status on frameshifting. Hypomodi®cation of tRNA Asn had only a slight effect on frameshifting and of the tRNA Lys mutants tested, only trmE (anticodon s 2 UUU) had a marked in¯uence, increasing the ef®ciency of the process at the slippery sequence UUUAAAA.
Sequence requirements for IBV frameshifting in E. coli
The ef®ciency of frameshift signals of the IBV type in the eukaryotic rabbit reticulocyte lysate (RRL) in vitro translation system has been shown to be in¯uenced by the nature of the slippery sequence, the integrity of the downstream RNA structure and the precise spacing between the two elements (Brierley et al., 1991 (Brierley et al., , 1992 . We began by con®rming that these requirements were maintained in E. coli. Complementary DNAs (cDNAs) containing variants of the IBV signal were subcloned (see Materials and Methods) into the E. coli expression vector pET3xc (Studier et al., 1990) to create the pMM series of plasmids (see Figure 1 ). pET3xc contains the ®rst 783 bp of coding sequence of the bacteriophage T7 gene 10,¯anked by a T7 promoter and transcription termination signal. In E. coli BL21 cells, which contain an IPTG-inducible T7 RNA polymerase, a 261 amino acid residue portion of gene 10 is expressed from pET3xc as an abundant, Triton-insoluble product that is relatively easy to purify (see Materials and Methods). In the pMM plasmids, the IBV cDNAs were cloned in frame with and downstream of the gene 10 sequence of pET3xc at a unique BamHI site. In BL21 cells, these constructs were predicted to express a 33 kDa non-frameshifted product corresponding to ribosomes that terminated at the IBV 1a stop codon and a 50 kDa frameshift product from ribosomes which frameshifted prior to encountering the stop codon and continued to translate the 1b ORF in the À1 frame. The constructs tested are shown in Figure 2 , the expressions in Figure 3 and the results are summarised in Table 2 . For the pET3xc plasmid, which does not contain an IBV insertion, only two major species were present, the N-terminal portion of gene 10, with an apparent molecular mass of about 34 kDa and lysozyme, carried over from the puri®cation procedure (see Materials and Methods). For the pMM expressions, three species were seen; lysozyme, a 36 kDa species corresponding to the non-frameshifted product and for most constructs, a 50 kDa frameshift product. The identity of the 36 kDa and 50 kDa proteins was con®rmed by demonstrating that both proteins reacted in Western blots with a monoclonal antibody directed against the N terminus of gene 10 (AMS Biotechnology Ltd, Europe; data not shown). We also con®rmed that the 36 kDa and 50 kDa proteins were restricted to the insoluble fraction of E. coli cell extracts; no soluble non-frameshifted or frameshifted protein was detected (data not shown). Figure 1 . The basic plasmids used in this study, the pMM series, were prepared by subcloning 585 bp NheI-EcoRI fragments containing the IBV frameshift region from plasmids pFS7, pFS8 or mutant derivatives (Brierley et al., 1989 (Brierley et al., , 1991 into BamHI-cleaved, plasmid pET3xc (Studier et al., 1990) . Both fragment(s) and vector were end-®lled using the Klenow fragment of DNA polymerase I prior to ligation with T4 DNA ligase. The resulting plasmids contain the IBV ORF 1a/1b frameshift signal (sequence information from base-pairs 12,286 to 12,511; Boursnell et al., 1987) ¯anked by portions of the in¯uenza A/PR8/34 PB1 gene (sequence information from base-pairs 1140 to 1167 (5 H ) and 1167 to 1500 (3 H ); Young et al., 1983 ) located downstream of, and in frame with, the ®rst 783 bp of coding sequence of the bacteriophage T7 gene 10. The ensemble is under the control of the bacteriophage T7 promoter and a T7 transcription termination signal is present at the end of the coding sequences. In the relevant T7-expressing bacteria (see Materials and Methods), the constructs are predicted to express a 33 kDa non-frameshifted product, corresponding to ribosomes that terminate at the IBV 1a stop codon and a 50 kDa frameshift product from ribosomes that frameshift prior to encountering the stop codon and continue to translate the 1b ORF in the À1 frame.
Figure 2. Frameshift constructs tested in E. coli. A, Slippery sequence variants. In each construct, the slippery sequence is boxed. Nucleotides that differ from the wild-type sequence (pMM2) are indicated in bold. In pMM10 and pMM11, the distance between the slippery sequence and RNA pseudoknot was decreased (GGG deleted) or increased (UAC inserted) by 3 nt, respectively. In pMM6 and pMM5, the pseudoknot was deleted (as in construct pFS7.6; Brierley et al., 1989) . B, Pseudoknot variants. Complementary and compensatory changes were created within the pseudoknot region. In this representation of the pseudoknot, the stems are arranged vertically and the loops are shown as thick lines. For each base-pair(s) studied, the two complementary changes (no base-pairing) and the compensatory change (base-pairing restored) are boxed and labelled with a mutant number. C. Stem ±loop constructs tested. Two constructs were tested that formed a stem± loop structure rather than a pseudoknot. Plasmid pMM23 forms only stem 1 due to a deletion that removed the downstream pseudoknot-forming region (as in construct pFS7.6; Brierley et al., 1989) . Plasmid pMM7 is a stem±loop construct in which the stem nucleotides are of the same length and nucleotide composition as the stacked stems of the pseudoknot in pMM2 (as in construct pFS8.26; Brierley et al., 1991) .
The generality of the simultaneous slippage model of frameshifting for sites expressed in E. coli is not fully established. It was important, therefore, to determine whether frameshifting at the IBV slippery sequence in E. coli deviated from the conventional simultaneous slippage mechanism ascertained in RRL. We created a series of mutations at or around the IBV slippery sequence and tested for frameshifting by expression in E. coli BL21 (see Figure 3A ). The wild-type IBV slippery sequence, UUUAAAC (pMM1), decoded by tRNA Asn (anticodon 5 H QUU 3 H ) stimulated only low-levels of frameshifting (2%), as did UUUAAAU (pMM8, 3%). In E. coli, therefore, tRNA Asn is considerably less slippery than it is in eukaryotic cells or the RRL (Brierley et al., 1989) . In contrast, tRNA Lys (anticodon 5 H U 8 UU 3 H ; where U 8 is mnm 5 s 2 U), which reads UUUAAAA (pMM9) and UUUAAAG (pMM2), was highly slippery; frameshifting at these sites occurring with great ef®ciency (15% and 40%, respectively). The slipperiness of E. coli tRNA Lys has been documented and will be discussed in detail later. In E. coli the hierarchy of frameshifting for the seventh nucleotide of the slippery sequence (UUUAAAN) was N G > A4U C. This is almost the reverse of the situation seen in RRL, where the hierarchy for N is C > A U4G (Brierley et al., 1992) , and is consistent with earlier studies of frameshifting in E. coli (Weiss et al., 1989; Tsuchihashi & Brown, 1992) . The introduction of different termination codons (UGA, pMM24; UAG, pMM27; UAA, pMM28) or an alternative sense codon (UGG, pMM25) immediately downstream of the slippery sequence had little effect on frameshifting, although a discernible reduction was seen with the UAG (32%) and UGA (29%) terminators. By¯anking the slippery sequence with termination codons (pMM26), one immediately downstream (UGA) in the zero phase and a second immediately upstream (UAA) in the À1 phase, we were able to con®rm that frameshifting takes place within the UUUAAAG stretch ( Figure 3B ). As with pMM27, we observed a slight reduction in frameshift ef®ciency with this construct, which had UAG as the downstream termination codon (see Discussion). We also introduced mutations within the slippery sequence. Unsurprisingly, the sequence UUUA-CAG (pMM30) was non-functional, since his mutation would prevent slippage of the A-site decoding tRNA. However a P-site mutation, UCUAAAG (pMM29) was fully competent in frameshifting, suggesting that in the case of the IBV signal in E. coli, the process does not involve simultaneous slippage of two tRNAs, but rather À1 slippage of a single tRNA Lys (from AAG to AAA). Puri®ed proteins expressed from the relevant frameshift plasmids were separated on SDS 15% polyacrylamide gels and detected by staining with Coomassie brilliant blue R as described in Materials and Methods. The nonframeshifted (stop) and frameshifted (frameshift or fs) products are indicated by arrows. Lysozyme (lyso) carried over from the puri®cation procedure is indicated by an arrow. The relevant mutant number is indicated at the bottom of each track. A number of abbreviations are employed: MW, high molecular mass protein size standards (Amersham); UUUAAAX, variant slippery sequence where X is A, C, G or U as indicated above the relevant track; spacer, the slippery sequence-pseudoknot spacing distance was increased (3 nt) or decreased (À3 nt) as indicated; no PK, pseudoknot deletion mutants with slippery sequence UUUAAAC (C) or UUUAAAG (G); stem 1 or stem 2 complementary (M) and compensatory (pWT) mutations were analysed in blocks of six (6 bp change), three (3 bp change) or single base-pairs (1 bp change); ssGGG, slippery sequence (UUUAAAG) and downstream codon are indicated; UAAssUAG, slippery sequence¯anked by termination codons; point, single point mutation in stem 2 of construct pMM4; only S1, construct can form only stem 1; simple-stem, construct forming long stem±loop structure as detailed in Figure 2C .
Ef®cient frameshifting at the IBV signal, at least in the RRL, depends upon the RNA pseudoknot structure, which cannot be replaced functionally by a stable stem ±loop structure of the same predicted size and nucleotide composition as the stacked stems of the pseudoknot (Brierley et al., 1991) . We investigated whether the RNA secondary structure requirements for frameshifting in E. coli were conserved by measuring the frameshift ef®ciency of a series of pseudoknot mutants (see Figures 2 and 3) . Complete removal of the pseudoknot dramatically reduced frameshifting, irrespective of whether the slippery sequence was UUUAAAC (pMM6) or UUUAAAG (pMM5). The pattern of frameshifting observed for mutations within the pseudoknot closely paralleled that seen in the RRL, in that destabilization of either stem of the pseudoknot reduced frameshifting ef®ciency (pMM3, 12, 13, 15, 20, 21 in stem 1; pMM4, 17, 18 in stem 2) and compensatory mutations predicted to restore the structure in general increased frameshifting (pMM16, 22 in stem 1; pMM19 in stem 2). Of the compensatory mutants in stem 1, two of the three analysed had frameshift ef®ciencies considerably below that seen for the wild-type structure (pMM14, 12% and pMM16, 20%). This is a phenomenon that has been observed in a number of studies of eukaryotic frameshifting (see ten Dam et al., 1995) and may reect a functional requirement for a particular pseudoknot conformation that is imprecisely reproduced in some of the compensatory mutants. The ef®ciencies of frameshifting measured for the stem 2 point mutations (pMM4, 15%; pMM17, 15% and pMM18, 9%) were considerably greater than that seen previously in RRL, where frameshifting in these mutants is reduced to about 5% (Brierley et al., 1991) . This supports the idea that ef®cient frameshifting in E. coli can be mediated by simple hairpin loop stimulators. Indeed, in construct pMM23, which contains the potential for formation of only stem 1, frameshifting occurred ef®ciently (22%). Moreover, when the pseudoknot of pMM2 was replaced by a large stem ± loop structure of the same predicted size and nucleotide composition as the stacked stems of the pseudoknot (pMM7), frameshifting occurred at a high level in E. coli cells (32%), in contrast to the situation in RRL, where such a structure promotes only low levels (1 to 2%) of frameshifting. This observation highlights an important difference between prokaryotic and eukaryotic ribosomes in terms of their response to RNA stimulators associated with frameshift sites. A similarity that is maintained, however, is the necessity for precise spacing between the stimulatory structure and the slippery sequence. As in the RRL (Brierley et al., 1989) , increasing (pMM11) or decreasing (pMM10) the spacing distance by three nucleotides greatly reduced frameshifting at the IBV site in E. coli.
To investigate the role of tRNA anticodon modi®cation in the frameshift process, pairs of plasmids with variations in the last nucleotide of the slippery sequence (UUUAAAN) were expressed in E. coli strains de®cient in tRNA modi®cation (see Table 1 ). In these experiments, T7 RNA polymerase was provided by infecting cells with bacteriophage l CE6 (see Materials and Methods), which contains the gene for T7 RNA polymerase under the control of the P L and P I promoters. Under these circumstances, we found that expression levels were generally lower than those seen upon IPTG-induction of BL21 cells and were in¯uenced by the growth-rate of the cells (re¯ecting the ef®ciency of the l CE6 infection). However, the signal to noise ratios (in terms of expressed proteins to cellular background) were suf®ciently tRNA Anticodon Modi®cation and Frameshifting high that estimates of frameshifting ef®ciency were reliable and reproducible. We studied two anticodon modi®cations, the Q base of tRNA Asn and the mnm 5 s 2 U base of tRNA Lys (see Figure 4 ).
Plasmids pMM1 (UUUAAAC) and pMM8 (UUUAAAU) were expressed in a wild-type host (SJ1512) and in two strains de®cient in the biosynthesis of Q due to a lack of functional tRNA guanine transglycosylase (TGT: SJ1515, K12 Átgt). In these strains, Q is not incorporated into tRNA and tRNA Asn has the anticodon 5 H GUU 3 H . As can be seen in Figure 5A , the absence of Q had only a modest effect on frameshifting, resulting in a twofold increase (UUUAAAU) or decrease (UUUAAAC) in frameshift ef®ciency (see Table 2 ). It is clear therefore that in E. coli, hypomodi®cation of the anticodon of tRNA Asn does not lead to a dramatic increase or decrease in frameshifting.
The role of mnm 5 s 2 U Unlike tRNA Asn , the anticodon wobble base of tRNA Lys is modi®ed at two positions, at position 5, where hydrogen is replaced by a methylaminomethyl group and at position 2, where oxygen is replaced by sulphur. Although a number of E. coli mutants exist that are defective in the synthesis of mnm 5 s 2 U, a fully unmodi®ed strain is not available. Plasmids pMM9 (UUUAAAA) and pMM2 (UUUAAAG) were expressed in wild-type hosts (TG1, TH48, DEV 1, TH79, TH160) and in the relevant defective strains listed in Table 1 . Essentially, four mutants were examined; trmC1 (Hagervall & Bjork, 1984) , which has the hypermodi®cation 5-carboxymethylaminomethyl-2-thiouridine (cmnm 5 s 2 U; TH69); trmC2 (Hagervall & Bjork, 1984) , containing the undermodi®ed 5aminomethyl-2-thiouridine (nm 5 s 2 U; TH49); trmE (Elseviers et al., 1984) , which possesses only the 2thiouridine substitution (s 2 U; TH78, DEV 16) and asuE107, which contains only the 5-methylaminomethyl substitution (mnm 5 U; TH159). In the trmC1, trmC2 and trmE strains, the presence of the sulphur atom was potentially problematic, since it can be replaced by selenium, which is thought to confer altered decoding properties (Wittwer & Table 1 ), the puri®ed expression products separated on SDS 15% polyacrylamide gels and detected by staining with Coomassie brilliant blue R as described in Materials and Methods. The non-frameshifted (stop) and frameshifted (frameshift) products are indicated by arrows. Lysozyme carried over from the puri®cation procedure is indicated by an arrow (lyso). The E. coli strain employed is indicated at the bottom of each track. The plasmids expressed were pMM1 (UUUAAAC), pMM2 (UUUAAAG), pMM8 (UUUAAAU) and pMM9 (UUUAAAA). The relevant genotype is indicated above each track (wt, wild-type with respect to the modi®cation genotype); Q; Q status, either absent (À) or present (). A, Bacteria cultured in LB medium; B, bacteria cultured in a de®ned minimal medium (see Materials and Methods), except asuE (LB medium). Long stem-loop 32 pMM12
Stem 1 3 bp change 8 pMM13
Stem 1 3 bp change 10 pMM14
Stem I pseudo-wt 12 pMM15
Stem 1 1 bp change 10 pMM3
Stem 1 1bp change 19 pMM16
Stem 1 pseudo-wt 20 pMM20
Stem 1 6 bp change 2 pMM21
Stem 1 6 bp change 2 pMM22
Stem 1 pseudo-wt 38 pMM4
Stem 2 point mutation 15 pMM17
Stem 2 1 bp change 15 pMM18
Stem 2 1 bp change 9 pMM19
Stem 2 Each value quoted represents the average of three to ®ve independent measurements, which varied by less than 5%; i.e. a measurement of 40% frameshift ef®ciency was between 38% and 42%. The abbreviations used are: PK, pseudoknot; wt, wild-type. Ching, 1989) . This was not a problem with the asuE strain; the level of mnm 5 Se 2 U has been measured in an asuE strain and was not detected (Kramer & Ames, 1988) . Previous studies, however, have indicated that selenium is not detectably incorporated into tRNA when bacteria are cultured in minimal medium (Wittwer & Stadtman, 1986) . Although these authors have suggested that this is also the case for LB medium, we decided to measure frameshifting in the trmC1, trmC2 and trmE strains during culture in a de®ned minimal medium prepared from chemicals of high purity in addition to assays in standard LB medium. The results of the analysis are shown in Figure 5 and are summarised in Table 2 . The signal to noise ratio in the minimal medium assays was increased somewhat as a consequence of the slower growth-rate of the strains. The asuE strain ( Figure 5B ) grew very slowly in minimal medium and was only tested in LB medium. The frameshift ef®ciencies measured for the various mutants were not in¯uenced by the growth medium. No effect on frameshifting was seen in the trmC1 or trmC2 strains when either the UUUAAAA or UUUAAAG-containing test plasmids were expressed. The most noticeable in¯uence was in the trmE strain, where frameshifting was stimulated over twofold (from 15% to 32%) at the UUUAAAA site and also increased at the UUUAAAG site, although to a lesser extent (from 40% to 48%). These increases were seen in two independent trmE strains (TH78, DEV 16). Frameshifting in the asuE background was unaltered at the UUUAAAA site but was reduced a little at the UUUAAAG site (from 40% to 33%).
The generality of the simultaneous slippage model of frameshifting for sites expressed in E. coli is not fully established. Weiss et al. (1989) have expressed a variant of the MMTV frameshift signal in E. coli and have shown that ribosomes respond to both of the tandem slippery codons of the MMTV frameshift signal as predicted by the simultaneous slippage model. At the frameshift signal of the E. coli dnaX gene, mutagenesis of the slippery sequence (A-AAA-AAG) has con®rmed that both lysine codons are required for ef®cient frameshifting (Tsuchihashi & Brown, 1992) . Similarly, frameshifting at the G-T ORF overlap required to produce a bacteriophage l tail assembly protein occurs by a two tRNA slip (Levin et al., 1993) . However, in the E. coli insertion element IS1, frameshifting is known to occur by À1 slippage of a single lysyl tRNA at the sequence A-AAA (from the underlined codon onto the overlapping AAA codon), despite the fact that the A 4 stretch is embedded within two potential and conventional slippery sequences (U-UUA-AAA-AAC; Sekine & Ohtsubo, 1992 ). An unexpected ®nding from our analysis of the slippery sequence requirements for IBV frameshifting in E. coli was the high ef®ciency of the UCUAAAG mutant (pMM29), since such a mutant has only low activity in RRL (Brierley et al., 1989) . The simplest explanation for this observation is that frameshifting at the IBV site in E. coli occurs by slippage of a single tRNA at the second homopolymeric triplet of the slippery sequence (UUUAAAX) rather than by simultaneous slippage of two tRNAs. The P-site codon in the mutant, UCUAAAG, is probably decoded by a minor tRNA Leu isoacceptor with anticodon 5 H UAG 3 H (Inokuchi & Yamao, 1995) . If this tRNA were to slip into the À1 reading frame (in accordance with the simultaneous slippage model) it could form only a single G-U base-pair with the À1 frame codon. At present, therefore, we favour single-tRNA slippage. Whether tRNA Lys slips when in the P or A-site of the ribosome is not know. Recent evidence supports the idea that frameshifting at the HIV-1 slippery sequence (U-UUU-UUA) in E. coli occurs not by simultaneous slippage of P and A-site-bound tRNAs, but when these tRNAs are in the ribosomal E and P-sites (Hors®eld et al., 1995) . This hypothesis was proposed following the discovery that the presence of a termination codon immediately downstream of the U 6 A stretch reduced frameshifting some ®ve-to tenfold in a manner that was independent of sequence context and could be modulated by prokaryotic release factor 2. These observations were consistent with the last six nucleotides of the slippery sequence occupying the E and P-sites and the termination codon the A-site prior to tRNA slippage. In the present study, we did detect a slight reduction in frameshift ef®ciency when the downstream terminators UAG and UGA were employed, raising the possibility that a fraction of the frameshift events monitored involve the E-site; but the data are most consistent with the slippery sequence occupying the standard P and A-sites. This conclusion is supported by the fact that, as is also seen in RRL (Brierley et al., 1989 (Brierley et al., , 1992 , the spacing distance between the slippery sequence and pseudoknot had to be maintained at six nucleotides for ef®cient frameshifting to occur. Protein sequencing studies and further mutagenesis experiments are in progress in an attempt to improve our understanding of the precise mechanism of tRNA slippage at the IBV site in E. coli.
The requirements for downstream RNA secondary structure were tested in constructs with the most ef®cient (UUUAAAG) slippery sequence, using a series of complementary and compensatory pseudoknot mutants. The response of prokaryotic ribosomes to these mutants was generally similar to that seen with the eukaryotic ribosomes of the RRL in vitro translation system (Brierley et al., 1991) . However, an important difference was noted; when the IBV pseudoknot was replaced by a stable stem ± loop structure of the same predicted size and nucleotide composition, frameshifting was maintained at a high level in E. coli cells, in con-trast to the situation in RRL, where such a structure promotes only low levels of frameshifting. In naturally occurring frameshift sites in E. coli, the requirements for stimulatory RNA structures appear to be variable. At the G-T ORF overlap of bacteriophage l, no stimulatory secondary structure is apparent (Levin et al., 1993) . In contrast, the dnaX frameshift signal includes both a downstream stem±loop (Tsuchihashi & Kornberg, 1990) and an upstream stimulatory element formed between a Shine-Delgarno-like sequence (5 H AGGGaG 3 H ) located 10 nt upstream of the slippery sequence and the 3 H -end of 16 S rRNA (3 H UCCUcC 5 H : Larsen et al., 1994) . Variation is also evident at the frameshift sites of bacterial insertion sequences (Chandler & Fayet, 1993) . In IS3, a pseudoknot is required for ef®cient frameshifting (Sekine et al., 1994) and in IS150, stimulation is via a downstream stem±loop that may form a pseudoknot (Vo È gele et al., 1991) . In contrast, although a number of potential RNA structures are predicted to form downstream of the slippery sequence of IS1 (Sekine & Ohtsubo, 1989; Escoubas et al., 1991) , they do not appear to play a role in frameshifting (Sekine & Ohtsubo, 1992) . Whatever the case, a stimulatory structure is absolutely required for IBV frameshifting in E. coli; the UUUAAAG sequence alone was unable to stimulate detectable frameshifting. This is perhaps unsurprising, since in the expression constructs employed, no obvious Shine-Delgarno-like sequences are present upstream of the slippery sequence.
Our investigation of the role of tRNA anticodon modi®cation in frameshifting was prompted by the experiments reported by Hat®eld et al. (1989 Hat®eld et al. ( , 1992 , who proposed that hypomodi®cation of the anticodons of those tRNAs implicated in decoding frameshift sites may promote ef®cient frameshifting. We began by expressing our frameshift reporter constructs in E. coli tgt mutants unable to biosynthesize Q. In these cells, however, we detected only a modest in¯uence of hypomodi®ed tRNA Asn , with frameshifting reduced by about twofold on the slippery sequence UUUAAAC and increased by a similar magnitude at the UUUAAAU site. So for tRNA Asn , at least in E. coli, anticodon hypomo-di®cation per se is insuf®cient to promote highly ef-®cient frameshifting. The modest effects noted, however, are consistent with the view that the strength of the pre-slippage codon-anticodon interaction is important in frameshifting. The hypomo-di®ed variant of tRNA Asn with anticodon 5 H GUU 3 H would be expected to pair more strongly with the AAC codon than with AAU, and hence frameshifting should decrease at the AAC codon and increase at AAU, as was observed. The low levels of frameshifting seen in E. coli at slippery sequences decoded by tRNA Asn , with or without Q, is in contrast to the situation in higher eukaryotic cells, where tRNA Asn is highly slippery (Brierley et al., 1992) , despite possessing an identical anticodon loop (Chen & Roe, 1978) . The altered frameshift capacity may simply be a re¯ection of the eukaryotic translational environment, but a role for Q in eukaryotic frameshifting cannot be ruled out.
The situation with UUUAAAA and UUUAAAG, decoded by tRNA Lys , is more complex, since the wobble base of this tRNA (mnm 5 s 2 U) is modi®ed at two positions (see Figure 4) and an E. coli strain expressing a fully unmodi®ed tRNA is unavailable. In the present study, of the four available mutants with altered modi®cation of the wobble base of tRNA Lys , only trmE (wobble base is 2-thiouridine) had a marked in¯uence on frameshifting, increasing the ef®ciency of the process over twofold at UUUAAAA and to a lesser extent at UUUAAAG. The asuE mutant (wobble base mnm 5 U) showed a small reduction in frameshifting at UUUAAAG. The increased ef®ciency seen in the trmE strains is consistent with the idea that hypomodi®cation of the anticodon stimulates frameshifting (Hat®eld et al., 1992) . However, this hypothesis cannot explain the intrinsic slipperiness of the hypermodi-®ed``wild-type'' tRNA Lys . The biological role (or at least one of the roles) of the tRNA Lys modi®cations is thought to be in the regulation of the confor-mational¯exibility or rigidity of the base to ensure the correct translation of codons during protein synthesis, particularly to prevent decoding of AAC and AAU. Proton NMR studies of the conformational characteristics of modi®ed uridine bases (Yokoyama et al., 1985) indicate that the mnm 5 s 2 modi®cation introduces conformational rigidity such that the ribose exclusively adopts a C3 H -endo form, which favours recognition of adenosine, allows weak recognition of guanosine and precludes binding to cytosine and uridine. In support of this, tRNA Lys has been shown to decode preferentially the AAA codon and has little af®nity for AAG (Lustig et al., 1981) . Tsuchihashi (1991) has rationalised the high-ef®ciency frameshift signal of the dnaX gene on the basis that the slippery sequence, AAAAAAG, is poorly recognised by tRNA Lys , which slips ef®ciently at the AAG codon due to a weak wobble base-pair. This concept is supported by the experiments reported by Tsuchihashi & Brown (1992) , who were able to inhibit frameshifting at the dnaX site by expressing an additional tRNA Lys with anticodon 5 H CUU 3 H . In these cells, frameshifting at the AAAAAAG slippery sequence is thought to have been prevented by the more stable recognition of the AAG codon by tRNA Lys CUU. We believe that the ef®cient frameshift observed at the IBV signal in E. coli (at UUUAAAG) is also a result of a restricted capacity of tRNA Lys to pair with AAG. The fact that the UUUAAAA slippery sequence is also highly permissive suggests that recognition of AAA by tRNA Lys is also unusual when compared, for example, with the decoding of AAC or AAU by tRNA Asn . Yokoyama et al. (1985) have suggested that both the 2 and 5-substituents contribute to the confor-tRNA Anticodon Modi®cation and Frameshifting mational rigidity of tRNA Lys , with the major input from the 2-thiocarbonyl group. The NMR studies by Agris and colleagues (Sierzputowska-Gracz et al., 1987; Agris et al., 1992) indicate a role for only the 2-position thiolation. A second interaction, a hydrogen bond, has been proposed, which forms between the amino group of 5 H -substitutents containing an aminomethyl moiety (mnm 5 and cmnm 5 ) and the 2 H -OH residue of the adjacent, un-modi®ed U33 base in the U-turn structure of the anticodon loop (Hillen et al., 1978; Yokoyama & Nishimura, 1995 ; and see Figure 4 ). Circular dichroism analysis of the tRNA suggests that the anticodon has an unusual conformation in which the wobble base is buried in the anticodon loop, possibly interacting via its 5 H -substituent with the N 6 threonylcarbamoyl (t 6 ) modi®cation of base A37 across the loop (Watanabe et al., 1993) . Support for this idea comes from studies of a chemically synthesised short oligoribonucleotide comprising U33mnm 5 s 2 U-U-U-t 6 A37 (cited by Agris, 1996) . In this doubly modi®ed pentamer, a unique interaction occurs between the two modi®cations that is thought to be between the amine group of mnm 5 and the amino acid residue of t 6 A, by hydrogen or ionic bonding. Furthermore, model-building studies predict that the anticodon domain U-turn in tRNA Lys is at the mnm 5 s 2 U34 base rather than at the usual invariant residue U33.
The structural studies described above suggest that the intrinsic shiftyness of tRNA Lys arises from its inability to recognise ef®ciently lysine codons as a consequence of the anticodon modi®cations, which are required to prevent erroneous decoding of asparagine codons. In this light, we expected that gross alterations in the modi®cation status (absence of thiolation in asuE, absence of the 5 H -substituent in trmE) would result in a tRNA that would be less restricted and perhaps pair more readily with lysine codons. Following this logic, frameshifting would be reduced in these mutants, since the codon-anticodon interaction would be more stable. Clearly this was not the case, and at present we are unable to explain these observations satisfactorily. The most likely explanation is that possession of either of the two modi®cations is suf®cient to reduce recognition of lysine codons and allow ef®cient frameshifting. The structural studies imply a role for the amino group of the 5 Hsubstituent of tRNA Lys , most likely in inter-loop contact with t 6 A37. The increase in frameshifting seen with trmE, which does not possess the amino group, may well re¯ect the loss of such an interaction, resulting in altered recognition of the AAA codon (and to a lesser extent the AAG codon) by the mutant tRNA. It seems unlikely that the effect seen with trmE is related to how well the mutant tRNA is aminoacetylated; ef®cient aminoacetylation appears to be correlated with the presence of the s 2 moiety in tRNAs of this class (Rogers et al., 1995) . The lack of effect of the trmC1 and trmC2 mutations, which retain the amino group, may suggest that the conformation of tRNA Lys in these strains is not suf®ciently different to in¯uence codon recognition. In the case of trmC1 (cmnm 5 s 2 U; TH69) and trmC2 (nm 5 s 2 U; TH49), model-building studies (Lim, 1994) support the idea that the modi®ed tRNAs would be expected to have similar decoding properties as the wildtype tRNA. Mutants defective in asuE, trmE and trmC all decrease the ef®ciency of the ochre suppressor tRNA supG, which is a derivative of tRNA Lys with anticodon 5 H mnm 5 s 2 UUA 3 H (reviewed by Bjo È rk, 1992) . Whether this re¯ects a reduced af®nity for the nonsense codon or another step in the suppression pathway is not clear, although the trmC1 mutation is known not to affect the binding properties of tRNA Lys to AAA or AAG programmed ribosomes (Elseviers et al., 1984) .
In conclusion, our data are not wholly consistent with either of the hypotheses advanced to explain the role of tRNA anticodon modi®cation in frameshifting. The observed effects of hypomodi®ed tRNA Asn on frameshifting at UUUAAAU/C are most consistent with the idea that the strength of the codon-anticodon interaction determines frameshift ef®ciency rather than being mediated primarily by hypomodi®ed tRNAs. However, the data obtained with variantly modi®ed tRNAs Lys are not readily explainable in terms of either model. Although the increased frameshifting seen in trmE strains is consistent with a role for hypomodi®ed tRNAs, the inherent slipperiness of the wild-type hypermodi®ed tRNA Lys argues strongly against this hypothesis. Nevertheless, the data obtained for the other tRNA Lys modi®cation mutants are dif®cult to interpret solely in terms of the predicted strength of codon-anticodon recognition. All ef®cient À1 frameshift sites in E. coli employ tRNA Lys as the A-site decoding tRNA and in all likelihood, this tRNA has a very unusual anticodon structure. In attempting to compare the role of tRNA anticodon modi®cation in frameshifting between prokaryotic and eukaryotic systems, we remain mindful that tRNA Lys may be a special case and that the role of modi®ed bases in eukaryotic frameshifting needs to be tested directly. Such studies are underway.
Site-directed mutagenesis was carried out by a procedure based on that of Kunkel (1985) as described (Brierley et al., 1989) . Mutants were identi®ed by dideoxy sequencing of single-stranded templates (Sanger et al., 1977) .
The starting plasmids pFS7, pFS8 or mutant derivatives (Brierley et al., 1989 (Brierley et al., , 1991 , which contain the IBV ORF 1a/1b frameshift signal¯anked by portions of the in¯uenza virus A/PR8/34 PB1 gene (Young et al., 1983) , were subjected to site-directed mutagenesis. In most cases, this was to change the IBV slippery sequence from UUUAAAC to UUUAAAG. Following mutagenesis, 585 bp NheI-EcoRI fragments encompassing the frameshift region were subcloned from the mutated plasmids into BamHI-cleaved pET3xc, an E. coli expression vector (Studier et al., 1990) . Both fragments and vector were end-®lled using the Klenow fragment of DNA polymerase I prior to ligation with phage T4 DNA ligase. The resulting plasmids, which comprise the pMM series, are shown in Figure 1 . The PB1:1a/1b:PB1 fragment is located downstream of, and in frame with, the ®rst 783 bp of coding sequence of the bacteriophage T7 gene 10 and the ensemble is under the control of the bacteriophage T7 promoter.
Bacteria were grown at 37 C in rich medium (LB; Maniatis et al., 1982) or in minimal salt medium (M9; Maniatis et al., 1982) containing thiamine (5 mg/l), glucose (0.2%, w/v) and the required amino acids (50 mg/l). The M9 medium was prepared using reagents of high purity (Aldrich Chemical Company). The genotypes and origins of the E. coli strains used are given in Table 1 . TH78 was constructed by transferring the trmE allele from DEV 16 into XA10B by phage P1 transduction, essentially according to Miller (1972) . Transductants were selected for valine resistance and co-transduction of trmE monitored by screening for an Arg À phenotype. In this screen, the presence of trmE was observed as an antisuppresor activity of supB, which suppresses poorly the amber mutation in argE. A lack of mnm 5 s 2 U and the presence of s 2 U in tRNA from TH78 was veri®ed by HPLC analysis according to Gehrke & Kuo (1990) . TH79 is an isogenic trmE strain. The isolation of the asuE107 mutation used in this study (TH159) will be described elsewhere. TH160 is an isogenic asuE strain. Transfer RNA puri®ed from TH159 was shown to contain mnm 5 U instead of mnm 5 s 2 U by combined liquid chromatography-mass spectrometry (LC/MS; unpublished results).
The sequence requirements for IBV frameshifting in E. coli were investigated by expressing the pMM plasmid series in E. coli BL21/DE3/pLysS cells ( Figure 1, Table 1 ), largely as described by Studier et al. (1990) . These cells contain, under the control of the lacUV5 promoter, the gene for T7 RNA polymerase inserted within the int gene of the prophage DE3, a l derivative. Expression of T7 RNA polymerase in BL21 cells can be induced by addition of isopropyl-b,D-thiogalactopyranoside (IPTG). Freshly transformed BL21 cells prepared by the method of Hanahan (1983) and containing plasmids of the pMM series were inoculated into 1.5 ml LB cultures containing ampicillin (50 mg/ml) and chloramphenicol (30 mg/ml). After three hours at 37 C, IPTG was added to 0.4 mM and incubation continued for a further three hours. Cells were pelleted, resuspended in 150 ml of lysis buffer (25 mM Tris (pH 8), 10 mM EDTA, 50 mM sucrose, 2 mg/ml lysozyme), placed on ice for 30 minutes and treated with DNase I (30 mg/ml) for a further 30 minutes at 37 C in the presence of 8 mM MgCl 2 and 1 mM MnCl 2 . Detergent solution (300 ml of 20 mM Tris (pH 7.5), 2 mM EDTA, 0.2 M NaCl, 1% (w/v) deoxycholic acid, 1% (v/v) Nonidet P-40) was added and the insoluble material harvested by centrifugation at 8000 g for two minutes. The pellet was washed three times with 0.5% (v/v) Triton X-100, 1 mM EDTA and the Triton-in-soluble material, which contained predominantly the pMM expression products, dissolved in 100 ml of sample buffer (Laemmli, 1970) . Aliquots were analysed on SDS/ 15% (w/v) polyacrylamide gels according to standard procedures (Hames, 1991) . Proteins were stained with Coomassie brilliant blue R (0.05%, w/v) in 10% (v/v) acetic acid, 50% (v/v) methanol, and destained in 10% acetic acid, 20% methanol. The relative abundance of non-frameshifted or frameshifted products was estimated (Adobe Photoshop and NIH Image software) by scanning densitometry and adjusted to take into account the differential size of the products. Scans were performed on gels whose proteins were stained to an intensity at the centre of the dynamic range of the scanner (Microtek IIXE Scanmaker). The frameshift ef®ciencies quoted in the text and summarised in Table 2 are the average of three to ®ve independent measurements that varied by less than 5%, i.e. a measurement of 40% frameshift ef®ciency was between 38% and 42%.
The in¯uence of tRNA anticodon modi®cation on IBV frameshifting was probed by expressing pMM plasmids in modi®cation-de®cient E. coli strains (Table 1) . Since the strains were non-lysogenic for DE3, expression of T7 RNA polymerase was achieved by infecting plasmidbearing cells with l CE6 (AMS Biotechnology UK Ltd.)> This phage contains the gene for T7 RNA polymerase under the control of the P L and P I promoters. Stocks of CE6 were prepared in E. coli LE392 by plate lysis (Maniatis et al., 1982) , collected by centrifugation (100,000 g for two hours) and resuspended at 10 12 pfu/ml. Modi®cation-de®cient strains harbouring pMM plasmids were grown until the absorbance at 600 nm was between 0.6 and 0.8, and infected with CE6 at 10 to 20 pfu/cell. After three hours, the cells were harvested and pMM expression products puri®ed and analysed as above.