Unstructured 5'-tails act through ribosome standby to override inhibitory structure at ribosome binding sites

Abstract

Initiation is the rate-limiting step in translation. It is well-known that stable structure at a ribosome binding site (RBS) impedes initiation. The ribosome standby model of de Smit and van Duin, based on studies of the MS2 phage coat cistron, proposed how high translation rates can be reconciled with stable, inhibitory structures at an RBS. Here, we revisited the coat protein system and assessed the translation efficiency from its sequestered RBS by introducing standby mutations. Further experiments with gfp reporter constructs assessed the effects of 5'-tails-as standby sites-with respect to length and sequence contributions. In particular, combining in vivo and in vitro assays, we can show that tails of CA-dinucleotide repeats-and to a lesser extent, AU-repeats-dramatically increase translation rates. Tails of increasing length reach maximal rate-enhancing effects at 16-18 nucleotides. These standby tails are single-stranded and do not exert their effect by structure changes in the neighboring RBS stem-loop. In vitro translation and toeprinting assays furthermore demonstrate that standby effects are exerted at the level of translation initiation. Finally, as expected, destabilizing mutations within the coat RBS indicate an interplay with the effects of standby tails.

Figure 1.

Experimental design and test of reporter system. (A) The DNA sequence encoding the sequestered RBS of the MS2 coat protein (red box) was translationally fused to gfp (green) either including (pMS2) or lacking (pMS2–0) the relatively unstructured flanking regions. The coat SD sequence and start codon are boxed (black). Grey squares indicate restriction sites. Red and black arrows indicate inverted repeat sequences. (B) Expression levels of gfp as detected by Western blot. Detection of GroEL was used as a loading control. This gel is representative of five replicates. (C) Expression levels of gfp as detected by plate reader. Fluorescence was measured as in Materials and Methods. Growth curves were similar between strains. The data are averages of three biological replicates. Error bars indicate the standard error.

Figure 2.

RNA segments flanking the coat RBS stem–loop contribute to standby activity. Expression of gfp was assessed by fluorescence measurements in cells carrying various deletion plasmids (Materials and Methods). Inserts had flanking segments deleted as shown schematically, either upstream (A), downstream (B), or on either side of the RBS stem–loop (C). Experiments were done in biological triplicates, and error bars show the standard error.

Figure 3.

Short sequences in front of the inhibitory RBS affect translation rates. The DNA sequence of the coat RBS stem–loop (in red) was translationally fused to gfp (green). Short sequences were inserted upstream of the stem–loop, immediately downstream of two A residues (positions +1 and +2 in mRNA). These were mono- or dinucleotide repeats of six or eight nucleotides (A), increasing numbers of (AU)-repeats (B) or of (CA)-repeats (C). Fluorescence levels are shown relative to that of the reference strain carrying pMS2 (see Figure 1), with error bars for three biological replicates.

Figure 4.

Addition of CA-repeats does not change the secondary structure of the coat RBS stem–loop. (A) Enzymatic and chemical structure probing was conducted on control mRNA (00) or mRNAs with tails of 4, 6 or 8 (CA)-repeats, (see Materials and Methods), as indicated. The mRNAs were mock-treated (lanes ‘–’), partially digested with double-strand-specific RNase V1 (V1), or treated with lead(II) acetate (Pb²⁺). UCGA: sequencing reactions on (CA)8 mRNA. The position of the SD and AUG start codon are indicated by red boxes. Regions of reactivity toward RNase V1 (red solid line) and lead(II) acetate (red dashed line) are indicated on the autoradiogram. (B) The localization of RNase V1 (filled triangles) and lead(II) acetate (black dots) cuts are shown on the secondary structure of the 5′-segment of (CA)8 mRNA. Black boxes: SD and AUG.

Figure 5.

Western analysis of translation rates on mRNAs with AU- and CA-tails in vivo and in vitro. GFP levels were monitored by Western analysis either of total protein (in vivo) extracted from cells carrying plasmids of the AU-series (A) and CA-series (C), or after in vitro translation of purified mRNAs from the AU-series (B) and CA-series (D). Detection used an antibody against GFP, and subsequently an anti-GroEL antibody (loading control). Arrows show the position of the longer GFP fusion protein (from pMS2), and all other GFP products (same size). The expanded box in (D) shows a longer exposure of this part of the gel, since the output from the CA-series was much stronger (see Results). Asterisks indicate unspecific antibody binding to proteins in the assay mix.

Figure 6.

The 5′ standby tails need to be single-stranded to promote translation and initiation complex formation. (A) The effect of blocking of the (CA)6 and (CA)8 standby tail by an antisense oligo (at 6.7 times molar excess), or a control oligo, was assayed by in vitro translation. The mRNAs used are indicated, and GFP was detected by Western blot. (B) Formation of a heteroduplex between the antisense oligo and the standby tail of (CA)8 mRNA was tested by RNase H cleavage (Materials and Methods). Cleavage was observed after reverse transcription using a 5′-labeled oligo annealed downstream. Cleavage near the base of the stem is observed only in the presence of the antisense oligo and RNase H (far right lane). (C) The toeprint experiment was conducted on several mRNA variants, with 30S and tRNA^fMet, with or without antisense or control oligo (Materials and Methods), as indicated. The position of the characteristic toeprint at +15 is shown. UAGC shows a sequencing ladder for reference. The gel is representative of three technical replicates.

Figure 7.

Effects of destabilizing the inhibitory RBS stem–loop. (A) Structure predictions for the coat RBS stem–loop and the two mutant variants. Calculated ΔG° values are shown (acc. to reference (62) version 3.5 at 37°C). (B) Western blot showing the effects of stem–loop destabilization in the absence or presence of standby tails in vivo. GroEL served as loading control. (C) Relative gfp expression obtained with the same strains as in (B) measured in the microplate reader. Error bars are given as the standard error for three biological replicates.