Transcription termination in vitro by bacteriophage T7 RNA polymerase. The role of sequence elements within and surrounding a rho-independent transcription terminator.
rho-Independent transcription terminators in Escherichia coli contain a dG+dC-rich dyad-symmetrical structure that encodes an RNA hairpin structure and an adjacent, downstream dA+dT-rich region which encodes uridines at the 3'-end of the transcript. In the threonine (thr) attenuator, there are at least six sequence segments in the DNA that might affect termination: the sequence upstream of the attenuator, the deoxythymidine-rich stretch immediately preceding the G+C-rich region, the G+C-rich region itself and its hairpin loop-encoding region, the deoxyadenosine tract following the G+C-rich region, and the following downstream sequence. Our previous studies (Jeng, S.-T., Gardner, J.F., and Gumport, R.I. (1990) J. Biol. Chem. 265, 3823-3830) indicate that both the stability and sequence of the RNA hairpin formed by the G+C-rich region and the length of the uridine tract encoded by the deoxyadenosine stretch influence the termination of T7 RNA polymerase in vitro. In this report, we demonstrate that the template deoxythymidine run upstream of the G+C-rich region, the loop-encoding segment, and the sequences upstream and downstream of the thr attenuator also affect termination. These results indicate that: 1) a deoxythymidine tract is not absolutely required for termination, but increasing the number of deoxythymidines from one to nine base pairs causes T7 RNA polymerase to terminate more efficiently; 2) a template with the natural loop sequence reversed results in a higher termination efficiency than one encoded by the the wild-type attenuator; 3) the termination of T7 RNA polymerase is affected by sequences both proximal and distal to the thr attenuator.
Jeng
S T
ST
Department of Biochemistry, University of Illinois, Urbana 61801.
Gardner
J F
JF
Gumport
R I
RI
D001483:Base Sequence; D004252:DNA Mutational Analysis; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D008969:Molecular Sequence Data; D010957:Plasmids; D012234:Rho Factor / Q000378:metabolism; D013329:Structure-Activity Relationship; D013604:T-Phages / Q000201:enzymology*; D013698:Templates, Genetic; D013728:Terminator Regions, Genetic; D014158:Transcription, Genetic; D014764:Viral Proteins
D012234:Rho Factor; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases