Programmed translational -1 frameshifting on hexanucleotide motifs and the wobble properties of tRNAs

Abstract

Programmed -1 ribosomal frameshifting, involving tRNA re-pairing from an AAG codon to an AAA codon, has been reported to occur at the sequences CGA AAG and CAA AAG. In this study, using the recoding region of insertion sequence IS3, we have investigated the influence on frameshifting in Escherichia coli of the first codon of this type of motif by changing it to all other NNA codons. Two classes of NNA codons were distinguished, depending on whether they favor or limit frameshifting. Their degree of shiftiness is correlated with wobble propensity, and base 34 modification, of their decoding tRNAs. A more flexible anticodon loop very likely makes the tRNAs with extended wobble more prone to liberate the third codon base, A, for re-pairing of tRNALys in the -1 frame.

Fig. 1. The IS3 frameshift region, its various derivatives and the plasmid reporter system. (A) The segment of IS3 shown and the derived mutants were cloned in the pOFX302 plasmid. (B) Frameshifting efficiency was determined by protein labeling with [³⁵S]methionine. The results obtained with two control strains (one with an in-phase construct, giving the theoretical 100% frameshifting value, and the other containing the vector plasmid, 0% frameshifting value), and with the IS3 ‘wild type’ region (wt) are shown. One culture of the vector- containing strain was labeled in the absence of IPTG (ni). The position of the product from normal translation (G10) or frameshifting (FS) is indicated. The calculated level of frameshifting and the 95% confidence interval (c.i.) are indicated below the relevant lanes. In the natural IS3 frameshift region an AUG codon (frame –1) overlapping the UGA stop codon of orfA (frame 0) is used to initiate synthesis of the OrfB protein. This AUG codon was changed to CUG in order to prevent initiation without interfering with frameshifting (Sekine et al., 1994).

Fig. 2. Effect on frameshifting of variants of the codon 3′ of the IS3 shift site. The mutations indicated in Figure 1A were introduced on the 3′side (nucleotides N₇N₈N₉) of the A AAG shift site and the modified frameshift regions were cloned into the pOFX302 reporter plasmid. (A) Summary of the results obtained by pulse-labeling of C CAA AAG N₇G₈N₉ constructs. (B) Results obtained by performing β-galactosidase assays with C GCA AAG N₇(G/A)₈N₉ constructs. The value for both motifs with GCC as 3′ codon is also given (wt stands for C CAA AAG).

Fig. 3. Effect on frameshifting of N₀N₁N₂A variants: correlation with wobble properties and modification of base 34 of the N₁N₂A-decoding tRNA (A and B), and variants where N₀ is identical to N₁ (C). In (A) and (C), frameshift efficiencies were measured by quantitation of [³⁵S]methionine labeled products. The error bars indicate the 95% confidence interval. In (B), the sequences of the anticodons of the E.coli NNA-decoding tRNAs including modifications of base 34 are shown. The modifications are abbreviated as follows: 5-methylaminomethyluridine (mnm⁵U), 5-methylaminomethyl-2-thiouridine (mnm⁵s²U), 5-caboxymethylaminomethyluridine (cmnm5U), 5-methoxyuridine (mo⁵U), uridine-5-oxyacetic acid (cmo⁵U), inosine (I) and lysidine (k²C). Three anticodon sequences are not from E.coli but from Bacillus subtilis (B.s.) or Mycoplasma capricolum (M.c.) as indicated.

Fig. 4. Frameshifting in modification-deficient mutants. Six NNA mutants (three low frameshifters and three high) were introduced into isogenic wild-type, aroD, mnmA and mnmE strains. Frameshifting was estimated by measuring the β-galactosidase activity of the resulting strains (in the absence of IPTG). The construct in which g10 and lacZ are in the same frame (in-phase construct in Figure 1) served to define the 100% reference activity and a construct in which the A AAG sequence was mutated to a non-slippery one (G AAA) was used to determine the background level.

Fig. 5. Models for frameshifting on hexameric motifs. The N₁N₂A₃ AAG hexamer is normally read as N₁N₂A₃ and A₄A₅G₆ in frame 0 (top). Base 34 of the N₁N₂A₃-decoding tRNA disengages from pairing with A₃ (stage 1). A-site tRNA^Lys re-pairs on the –1 frame A₃A₄A₅ codon with re-positioning of the mRNA (stage 2). In (A), the P-site anticodon irreversibly dissociates from the mRNA whereas in (B), anticodon bases 36 and 35 maintain pairing. Accommodation is then completed (stage 3), bringing the two tRNAs and the mRNA in the configuration seen in crystallized complexes (Yusupova et al., 2001) allowing peptidyl-transfer and standard three-base translocation establishing the change in frame (stage 4).