Transcriptional terminator is a positive regulatory element in the expression of the Escherichia coli crp gene

• Published in: The Journal of biological chemistry 1991-01, Vol.266 (3), p.1721-1727
• Main Authors: AIBA, H, HANAMURA, A, YAMANO, H
• Format: Article
• Language: English
• Subjects: Base Sequence; Biological and medical sciences; Blotting, Northern; Cloning, Molecular; DNA Mutational Analysis; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Bacterial; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Receptors, Cyclic AMP - genetics; Regulatory Sequences, Nucleic Acid; Restriction Mapping; RNA, Bacterial - metabolism; RNA, Messenger - metabolism; Terminator Regions, Genetic; Transcription. Transcription factor. Splicing. Rna processing
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(18)52355-3

Plasmids were constructed that contain deletions in the stem region of the presumed rho-independent terminator of the cloned crp gene of Escherichia coli. The level of cyclic AMP binding activity and the amount of CRP in cells harboring the deletion plasmids were found to be significantly lower than those in cells harboring the wild-type crp plasmid. Quantitative S1 assays indicated that the steady-state levels of crp mRNA were markedly reduced in cells harboring the deletion plasmids. Evidence was also presented to show that the crp mRNAs produced from deletion plasmids are less stable than that from the intact crp gene. In vitro transcription assays revealed that the putative crp terminator is indeed a rho-independent terminator. Using the galK expression system and Northern blot analysis we showed that the crp terminator is functional in vivo. Moreover it was shown that the deletion mutations in the stem region of the crp terminator cause a significant readthrough. We conclude that the 3'-flanking sequence of the crp gene acts to stabilize its own mRNA as well as to terminate transcription.