ABSTRACT
When Escherichia coli cells are shifted to low temperatures (e.g. 15 degrees C), growth halts while the 'cold shock response' (CSR) genes are induced, after which growth resumes. One CSR gene, pnp, encodes polynucleotide phosphorylase (PNPase), a 3'-exoribonuclease and component of the RNA degradosome. At 37 degrees C, ribonuclease III (RNase III, encoded by rnc) cleaves the pnp untranslated leader, whereupon PNPase represses its own translation by an unknown mechanism. Here, we show that PNPase cold-temperature induction involves several post-transcriptional events, all of which require the intact pnp mRNA leader. The bulk of induction results from reversal of autoregulation at a step subsequent to RNase III cleavage of the pnp leader. We also found that pnp translation occurs throughout cold-temperature adaptation, whereas lacZ(+) translation was delayed. This difference is striking, as both mRNAs are greatly stabilized upon the shift to 15 degrees C. However, unlike the lacZ(+) mRNA, which remains stable during adaptation, pnp mRNA decay accelerates. Together with other evidence, these results suggest that mRNA is generally stabilized upon a shift to cold temperatures, but that a CSR mRNA-specific decay process is initiated during adaptation.
Subject(s)
Adaptation, Biological/genetics , Cold Temperature , Escherichia coli/genetics , Polyribonucleotide Nucleotidyltransferase/genetics , Bacterial Proteins/genetics , Escherichia coli/enzymology , Exoribonucleases/metabolism , Gene Expression Regulation, Bacterial , Gene Expression Regulation, Enzymologic , Polyribonucleotide Nucleotidyltransferase/biosynthesis , RNA Processing, Post-Transcriptional , RNA Stability , RNA, Bacterial/biosynthesis , RNA, Messenger/biosynthesis , Untranslated RegionsABSTRACT
There are four transcriptional promoters present in the 5' control region of the Escherichia coli DNA topoisomerase I (topA) gene. These were identified with Bal31 nuclease-generated deletions and mapping of the 5' ends of the mRNAs with avian reverse transcriptase. Recombinant plasmids with all or some of these promoters fused to the galactokinase (galK) gene-coding region have been constructed and used to study transcription from the promoters both in vitro and in vivo. The promoter (P1) closest to the starting ATG codon has a near consensus -35 sequence (GTTGATA) but unusual -10 (CATATCG) sequence. The other three promoters (P2, P3 and P4) are clustered together 60 base-pairs further upstream. Negative DNA supercoiling is required for efficient transcription from P1, P1 + P2 + P3 + P4, P2 + P3 + P4, P3 + P4 and P4 alone. The combination of all four promoters demonstrates greater supercoiling dependence than does any of the other subsets tested.
