Release factor 2 frameshifting sites in different bacteria

Abstract

The mRNA encoding Escherichia coli polypeptide chain release factor 2 (RF2) has two partially overlapping reading frames. Synthesis of RF2 involves ribosomes shifting to the +1 reading frame at the end of the first open reading frame (ORF). Frameshifting serves an autoregulatory function. The RF2 gene sequences from the 86 additional bacterial species now available have been analyzed. Thirty percent of them have a single ORF and their expression does not require frameshifting. In the approximately 70% that utilize frameshifting, the sequence cassette responsible for frameshifting is highly conserved. In the E. coli RF2 gene, an internal Shine-Dalgarno (SD) sequence just before the shift site was shown earlier to be important for frameshifting. Mutagenic data presented here show that the spacer region between the SD sequence and the shift site influences frameshifting, and possible mechanisms are discussed. Internal translation initiation occurs at the shift site, but any functional role is obscure.

Fig. 1. Statistical comparative analysis of the RF2 frameshifting cassette (top panel) with the corresponding regions of RF2 mRNAs that do not utilize frameshifting for their expression (lower panel). Each column represents the frequency within a subset of bacterial RF2 genes of a particular nucleotide at the position corresponding to the given E. coli RF2 mRNA sequence. Yellow represents A; light blue, C; red, G; green, U. Particular elements within mRNA are indicated with a black line as follows: LPR, low purine region; SD, Shine–Dalgarno sequence; FS, frameshifting site; IIS, internal initiation site; TC, termination context.

Fig. 2. Analysis of the effect of nucleotide identity in the spacer region between the stimulatory SD sequence and the frameshift site. (A) Outline of genetic constructs. (B) PAGE separation of the pulse–chase-labeled lysates of E. coli expressing genetic constructs containing the RF2 frameshifting cassette with different spacers. K, non-induced cells; WT, frameshift cassette with wild-type spacer (UAU); sequences of other spacers are written above each lane. TERM, product of termination; FS, product of frameshifting. (C) Plot representing the efficiency of termination as calculated from several independent experiments. White boxes are error bars.

Fig. 3. Analysis of internal initiation in an RF2 frameshift cassette. (A) Outline of genetic constructs. (B) PAGE separation of expressed proteins after Ni–NTA isolation. II, product of internal initiation; FS, product of frameshifting; Ter, product of termination.