mRNA stability is regulated by a coding‐region element and the unique 5′ untranslated leader sequences of the three Synechococcus psbA transcripts

Abstract

SummaryThe psbAI and psbAIII transcripts in Synechococcus sp. strain PCC 7942 are subject to accelerated turnover when cells are exposed to high light intensities, but psbAII message stability is unaffected. We used a psbAI 'minigene' which has a part of the coding sequence removed as a reporter gene in order to identify the cis-acting elements of the transcript that determine stability. While engineering the minigene to optimally mimic the native gene, we identified a stabilizer element within the open reading frame, corresponding to the coding region for the first membrane span of the D1 protein, the presence of and translation through which was essential for normal psbA mRNA stability. We propose that this stabilizer is a site for ribosome pausing, and that accumulation of ribosomes on the transcript upstream of the pause site increases stability. To identify the elements that regulate the differential responses of the psbA transcripts to high-light growth, sequences from psbAII and psbAIII were substituted in the psbAI minigene reporter. The chimeric reporter transcripts established that the psbAI and psbAIII untranslated leaders determine the faster turnover of these messages. The untranslated leader regions of the psbA transcripts may regulate mRNA stability by modulating translation and thereby stability, or by recruiting RNA-binding proteins that affect mRNA turnover more directly.

Chloroplast Biogenesis of Photosystem II Cores Involves a Series of Assembly-Controlled Steps That Regulate Translation

“…Only when at least 60 nucleotides of the psbA coding sequence were added in frame, upstream of the reporter gene, did the chimeric mRNA accumulate, enabling the phototrophic growth of the transformants (data not shown). These results point to the existence of cis-acting element stabilizing psbA-driven transcripts within the coding sequence of psbA, as already described for other chloroplast genes in C. reinhardtii (Singh et al, 2001) or for psbA in cyanobacteria (Kulkarni and Golden, 1997). Thus, we used for the rest of our study transformed strains expressing cytochrome f translated under the control of the psbA 59UTR, followed by the 60 first nucleotides of the psbA coding sequence, hereafter called bAf because they express a 59psbA-driven cytochrome f.…”

Functional Elements of the Strong psbAI Promoter of Synechococcus elongatus PCC 7942

“…These responses can be triggered by changes in light fluence or quality and are independent of photosynthetic electron flow, invoking a genuine response to light rather than to redox changes (32,36). Information that targets these messages, but not that of psbAII, for accelerated degradation at high light resides within their untranslated leaders (22). The apparent half-life of loss of the psbAI transcript at high light is approximately equivalent to the half-life of the message in the presence of a transcription inhibitor, which implies that no new transcription contributes to the psbAI message pool under these conditions.…”

“…The psbAI transcript rapidly decreases in abundance when cells are shifted to high light, with an apparent half-life of about 10 to 12 min (22,23). This is, at least in part, attributable to destabilization of the transcript, which depends on the 52-nucleotide untranslated leader (22).…”

Roles for Sigma Factors in Global Circadian Regulation of the Cyanobacterial Genome

“…Fusions of Ptrc to rpoD3, rpoD4, and sigC were then cloned as BglII fragments into the NS1 vector pAM1303 to incorporate the overexpression constructs into the cyanobacterial genome (18). A 4-kb BstZ171-NgoMIV fragment bearing the rpoD2 coding region fused to the Ptrc promoter was cloned into the NS1 vector pAM2314 cut with XmaI and StuI.…”