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Journal of Bacteriology, October 1999, p. 6284-6291, Vol. 181, No. 20
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Mutation Analysis of the 5' Untranslated Region of the Cold Shock cspA mRNA of Escherichia coli

Kunitoshi Yamanaka, Masanori Mitta,dagger and Masayori Inouye*

Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854

Received 7 April 1999/Accepted 6 August 1999

The mRNA for CspA, a major cold shock protein in Escherichia coli, contains an unusually long (159 bases) 5' untranslated region (5'-UTR), and its stability has been shown to play a major role in cold shock induction of CspA. The 5'-UTR of the cspA mRNA has a negative effect on its expression at 37°C but has a positive effect upon cold shock. In this report, a series of cspA-lacZ fusions having a 26- to 32-base deletion in the 5'-UTR were constructed to examine the roles of specific regions within the 5'-UTR in cspA expression. It was found that none of the deletion mutations had significant effects on the stability of mRNA at both 37 and 15°C. However, two mutations (Delta 56-86 and Delta 86-117) caused a substantial increase of beta -galactosidase activity at 37°C, indicating that the deleted regions contain a negative cis element(s) for translation. A mutation (Delta 2-27) deleting the highly conserved cold box sequence had little effect on cold shock induction of beta -galactosidase. Interestingly, three mutations (Delta 28-55, Delta 86-117, and Delta 118-143) caused poor cold shock induction of beta -galactosidase. In particular, the Delta 118-143 mutation reduced the translation efficiency of the cspA mRNA to less than 10% of that of the wild-type construct. The deleted region contains a 13-base sequence named upstream box (bases 123 to 135), which is highly conserved in cspA, cspB, cspG, and cspI, and is located 11 bases upstream of the Shine-Dalgarno (SD) sequence. The upstream box might be another cis element involved in translation efficiency of the cspA mRNA in addition to the SD sequence and the downstream box sequence. The relationship between the mRNA secondary structure and translation efficiency is discussed.

* Corresponding author. Mailing address: Department of Biochemistry, Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854. Phone: (732) 235-4115. Fax: (732) 235-4559. E-mail: inouye{at}umdnj.edu.

dagger Present address: Takara Shuzo Co., Ltd., Biotechnology Research Laboratories, Seta 3-4-1, Otsu, Shiga 520-2193, Japan.

Journal of Bacteriology, October 1999, p. 6284-6291, Vol. 181, No. 20
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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