Expression of the E.coli hemolysin secretion gene hlyB involves transcript anti-termination within the hly operon.
The genes hly A and hly B dictating synthesis and secretion of hemolytic toxin by Escherichia coli are transcribed as part of an operon hly C, hly A, hly B but are separated by an inverted repeat and poly-T sequence characteristic of a rho-independent terminator. The hly A-hly B intergenic sequence caused a reduction of in vivo transcriptional read-through from the gal promotor into gal K when inserted in the pKG1900 terminator-probe vector and also in vitro 3' to the bacteriophage T7 luminal diameter 10 promotor. Hybridisation of mRNA generated by the hly determinant of recombinant DNA pBR202-312 to an antisense RNA probe spanning the hly intergenic sequence revealed specific termination of 80% of in vivo hly transcripts within the poly-T sequence, immediately preceding the hly B translation start. Extension of the hly determinant with 3.5kbp of hly promotor-proximal DNA sequence raised intracellular and cell-free hemolytic activity 3-fold and 20-fold respectively and increased markedly the secretion of the 107Kd hemolysin protein (HlyA). Parallel hybridisation of in vivo mRNA to hly A and hly B antisense probes revealed that levels of hly A and hly B mRNA were increased by approximately 3-fold and 90-fold. The dramatically enhanced level of hly B mRNA resulted primarily from specific suppression of transcription termination at the intergenic rho-independent terminator from 80% to 1%, thus allowing virtually all hly A transcripts to elongate into hly B.
Koronakis
V
V
Department of Pathology, Cambridge University, UK.
Cross
M
M
Hughes
C
C
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D005796:Genes; D005798:Genes, Bacterial; D005809:Genes, Regulator; D006460:Hemolysin Proteins; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009876:Operon; D012329:RNA, Bacterial / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013728:Terminator Regions, Genetic; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins; C036507:Hlya protein, E coli; D008565:Membrane Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins