Effect of the pufQ‐pufB intercistronic region on puf mRNA stability in Rhodobacter capsulatus

Abstract

Differential expression of genes localized within the polycistronic puf operon of Rhodobacter capsulatus is partly due to altered stabilities of individual mRNA segments. We show that the 5' untranslated region (UTR) of pufB contributes to the unusual longevity of the 0.5 kb light-harvesting (LH) I specific pufBA mRNA and of the 2.7 kb pufBALMX mRNA. Three stem-loop structures have been identified within the pufQ-pufB intercistronic region by means of RNA secondary-structure analysis in vitro and in vivo. Deletion analysis of the pufB 5' UTR indicates that the complete set of secondary structures is required to maintain wild-type levels of pufBA mRNA stability. A phylogenetic comparison of pufB 5' UTRs of other photosynthetic bacteria reveals an evolutionary conservation of the base-pairing potential despite sequence divergence. Comparison of puf mRNA decay in Escherichia coli strains with or without endoribonuclease E (RNase E) activity suggests that the pufB 5' secondary structures protect the downstream mRNA segment against degradation by RNase E. Removal of the 117-nucleotide pufQ-pufB intercistronic region results in loss of stability for the pufBA and pufBALMX mRNAs with concomitant stabilization of the full-length puf primary transcript (QBALMX). We therefore conclude that the deleted sequence functions both as a stabilizing element for pufBALMX and pufBA segments and as a target site for initial rate-limiting decay of the unstable pufQBALMX mRNA.

The aprX–lipA operon of Pseudomonas fluorescens B52: a molecular analysis of metalloprotease and lipase production The GenBank accession numbers for the sequences reported in this paper are AF216700, AF216701 and AF216702.

“…An alternative model for low-temperature regulation proposes that the decay rate of the lipase-encoding segment of the polycistronic messenger varies with temperature. Differential segmental decay rates have been observed in the case of the puf operon, and furthermore, are subject to regulation by oxygen (Klug, 1991 ;Heck et al, 1996 ;Rauhut & Klug, 1999). Stem-loop structures are known to influence mRNA decay and a number of these have been noted within the intergenic regions of the B52 operon (see Fig.…”

The influence of Hfq and ribonucleases on the stability of the small non-coding RNA OxyS and its targetrpoSinE. coliis growth phase dependent

“…The intensity of the sRNA and mRNA bands was normalized to the intensity of the 5S or 16S rRNA. Total RNA was isolated by hot-phenol extraction 42 and RNA was stored in 100 μl of 20 mM sodium phosphate (pH 6.5), 1 mM EDTA at -20°C. RNA samples (~30 μg) were denatured for 10 minutes at 65°C in loading buffer containing 50% deionized formamide, separated on urea-polyacrylamide (10%) gels or on a 1% (w/v) agarose 2.2 M formaldehyde gel, and transferred to nylon phase (data not shown).…”

Cloning and transcriptional analysis of the nifUHDK genes of Trichodesmium sp. IMS101 reveals stable nifD, nifDK and nifK transcripts

“…Processing of long mRNA by RNA-specific endonuclease( s) and exonuclease(s) has been described or suggested in E. coli and Rhodobacter capsulatus (Belasco et al, 1985 ;Heck et al, 1996;Peterson, 1992;Willison et al, 1993). In this study it was shown (Fig.…”