Transcription termination by bacteriophage T7 RNA polymerase at rho-independent terminators.
We have investigated the mechanism of transcription termination by T7 RNA polymerase using templates encoding variants of the transcription-termination structure (attenuator) of the regulatory region of the threonine (thr) operon of Escherichia coli. The thr attenuator comprises the following two distinct structural elements: a G + C-rich inverted repeat, which encodes an RNA hairpin structure, and A + T-rich regions, one of which contains a continuous sequence of template deoxyadenosine residues within which the transcription terminates. Fourteen attenuator variants were analyzed and we find that not only the hairpin structure itself but also its sequence influences termination. Furthermore, the formation of a hairpin in the RNA encoded by the A + T-rich regions of the attenuator is not mandatory for termination. A series of seven deletion variants that successively shorten the deoxyadenosine tract in the attenuator template were also analyzed. Results from these experiments indicate that complete readthrough occurs when there are four or fewer deoxyadenosine residues. With 5 template deoxyadenosine residues there is 5% termination increasing to 32% with 8 deoxyadenosines, the value produced by the wild-type attenuator. In addition, a comparison with E. coli RNA polymerase shows that T7 RNA polymerase requires a more perfect region of dyad symmetry and a longer deoxyadenosine tract than does the bacterial enzyme to terminate with maximum efficiency.
Department of Biochemistry, College of Medicine, University of Illinois, Urbana 61801.
S T
ST
Jeng
J F
JF
Gardner
R I
RI
Gumport
D001482:Base Composition; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D005809:Genes, Regulator; D005822:Genetic Vectors; D009876:Operon; D010957:Plasmids; D012234:Rho Factor / Q000378:metabolism*; D013604:T-Phages / Q000201:enzymology* / Q000235:genetics; D013698:Templates, Genetic; D013728:Terminator Regions, Genetic; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic
D012234:Rho Factor; D014157:Transcription Factors; D012321:DNA-Directed RNA Polymerases