Translation at higher than an optimal level interferes with coupling at an intercistronic junction

Abstract

In pairs of adjacent genes co‐transcribed on bacterial polycistronic mRNAs, translation of the first coding region frequently functions as a positive factor to couple translation to the distal coding region. Coupling efficiencies vary over a wide range, but synthesis of both gene products at similar levels is common. We report the results of characterizing an unusual gene pair, in which only about 1% of the translational activity from the upstream gene is transmitted to the distal gene. The inefficient coupling was unexpected because the upstream gene is highly translated, the distal initiation site has weak but intrinsic ability to bind ribosomes, and the AUG is only two nucleotides beyond the stop codon for the upstream gene. The genes are those in the filamentous phage IKe genome, which encode the abundant single‐stranded DNA binding protein (gene V) and the minor coat protein that caps one tip of the phage (gene VII). Here, we have used chimeras between the related phage IKe and f1 sequences to localize the region responsible for inefficient coupling. It mapped upstream from the intercistronic region containing the gene V stop codon and the gene VII initiation site, indicating that low coupling efficiency is associated with gene V. The basis for inefficient coupling emerged when coupling efficiency was found to increase as gene V translation was decreased below the high wild‐type level. This was achieved by lowering the rate of elongation and by decreasing the efficiency of suppression at an amber codon within the gene. Increasing the strength of the Shine–Dalgarno interaction with 16S rRNA at the gene VII start also increased coupling efficiency substantially. In this gene pair, upstream translation thus functions in an unprecedented way as a negative factor to limit downstream expression. We interpret the results as evidence that translation in excess of an optimal level in an upstream gene interferes with coupling in the intercistronic junction.

Translational Control of the SigR-Directed Oxidative Stress Response in Streptomyces via IF3-Mediated Repression of a Noncanonical GTC Start Codon

“…3A). As a consequence, it has been assumed that the two genes are translationally coupled (28, 29) to ensure that the two proteins are made in equimolar amounts and that all translation of rsrA would depend on the prior translation of sigR (2, 6, 25, 30). The fact that changing the sigR start codon to ATG caused a white (Spo − ) phenotype, as described above, implied that the translational coupling of sigR and rsrA is incomplete, such that the increase in SigR translation was not reflected in an equal increase in the level of RsrA translation (SigR > RsrA).…”

In‐frame overlapping genes: the challenges for regulating gene expression

“…This was provided by the set of su7 amber suppressors characterized by Yarus and colleagues (Yarus et al ., 1986; Schultz and Yarus, 1994). They span a wide range of efficiencies, and are present on compatible low‐copy plasmids under control of an IPTG‐inducible promoter (Yu et al ., 2001). The suppressors contain the wild‐type amber suppressor sequence or site‐directed base changes in the anticodon loop.…”

“…The work described here has established that in‐frame overlapping genes II and X are under two forms of translational control. The first arises from the natural interference that elongating ribosomes can impose on new initiation events within or at the ends of coding regions (Shaw and Murialdo, 1980; Berkhout et al ., 1985; Das and Yanofsky, 1989; Yu et al ., 2001). The second is provided by an extended RNA secondary structure conserved in the Ff filamentous phages which sequesters the gene X ribosome binding site in duplex RNA on both mRNAs encoding gene X.…”

Non‐canonical RNA arrangement in T4‐even phages: accommodated ribosome binding site at the gene 26‐25 intercistronic junction

“…The SD sequence is a prerequisite of translation driven by reinitiation (Andre et al ., 2000; Yu et al ., 2001). The 70S ribosome can bind the SD sequence without dissociation into subunits (Takahashi et al ., 2008).…”

“…The 70S ribosome can bind the SD sequence without dissociation into subunits (Takahashi et al ., 2008). Considering that reinitiation efficiency depends on how well the 30S or 70S maintains contact with the mRNA before a new initiation complex is assembled (Yu et al ., 2001), the relatively strong SD sequence GAGG in the 25 TIR is likely to be a significant factor increasing both independent translation initiation and reinitiation efficiency. This quantitative study shows that gene 25 expression is marginally dependent on the upstream gene 26 translation.…”