Deletion analysis of cspA of Escherichia coli: requirement of the AT‐rich UP element for cspA transcription and the downstream box in the coding region for its cold shock induction

Abstract

SummaryIn order to analyse the mechanism of cold shock induction of CspA, a major cold shock protein of Escherichia coli, deletion analysis of the cspA gene was carried out. It was found that (i) the AT-rich sequence (¹47 to ¹38) upstream of the cspA ¹35 region may act as the UP element playing a crucial role in cspA transcription at both 37ЊC and 15ЊC; (ii) the unusually long 5Ј-UTR of the cspA mRNA has negative effects on cspA expression at 37ЊC; and (iii) in contrast, the 5Ј-UTR exerts a positive effect on mRNA stabilization at low temperature. Furthermore, it was demonstrated that the 14 base downstream box (DB) locating 12 bases downstream of the initiation codon of the cspA mRNA and complementary to a region near the decoding region of 16S rRNA was essential for the mRNA translation during the growth lag acclimation phase immediately after cold shock. During this phase, translation of non-cold shock gene mRNAs is blocked, since they require cold shock-specific ribosomal factors for the formation of the translation initiation complex. It is proposed that DB in cold shock mRNAs allows the formation of a stable initiation complex at low temperature in the absence of the cold shock ribosomal factors.

The Cold Box Stem-loop Proximal to the 5′-End of theEscherichia coli cspA Gene Stabilizes Its mRNA at Low Temperature

“…pCB01Z to pCB09Z were constructed using the same primers and mutagenesis method as described above, except that plasmid pMM158 was used as a template. pMM158 contains a cspA-lacZ fusion gene and is identical to pMM67 (9) except that it has a longer promoter with the 5Ј-end extended to Ϫ158.…”

“…It has been shown that the cspA promoter is highly active at both high and low temperatures (8). An AT-rich upstream-element immediately upstream of the Ϫ35 region has been implicated to contribute to the strength of the cspA promoter (9). It has also been reported that the cspA promoter is modestly activated after cold shock (10,11).…”

Regulation of gene expression at low temperature: role of cold-inducible promoters

“…The 59 UTR has also been reported to be important for mRNA stabilization following cold shock (Mitta et al, 1997). The conservation of the long 59 UTR in low-temperature-regulated genes suggests strongly that their occurrence is more than a coincidence and supports their role in gene regulation.…”

“…The authors concluded that the optimal temperature ranges for induction of the four E. coli cold-shock-induced csp genes (cspA, B, G and I) were not the same, thereby indicating that whilst the 59 UTR and cold-box elements may be involved in regulation of gene expression, specific sequence differences in the 59 UTR and cold-box elements may play important roles in regulation. Thus, it is apparent that the 59 UTR plays an important role with respect to regulation of cold-inducible genes, but studies using cspA : : lacZ fusions, which contained a variety of deletions of the 59 UTR (Mitta et al, 1997;Yamanaka, 1999), indicated that deleting the cold-box had little effect on cold-shock induction of b-galactosidase activity and, instead, a region 11 bp upstream of the ribosome-binding site was important for translational efficiency of gene expression. These data indicate that the precise mechanism by which regulation occurs is unclear.…”

“…The genes that contain the cold-box element within the 59 UTR are regulated by cold shock, and appear to be controlled by a range of transcriptional and translational control mechanisms (reviewed by Graumann & Marahiel, 1998;Panoff et al, 1998;Phadtare et al, 1999;Thieringer et al, 1998;Yamanaka et al, 1998;Mitta et al, 1997). As noted above, the 59 UTRs in most vertebrates are ,100 bp, but are longer in genes that are tightly controlled (UhlmannSchiffler et al, 2002), and such structures can modulate translation by the formation of secondary structures and by RNA-protein interaction (Gray & Wickens, 1998;UhlmannSchiffler et al, 2002).…”