Bacteriophage SP6 RNA polymerase mutants with altered termination efficiency and elongation processivity.
An Escherichia coli strain containing two plasmids was developed for in vivo isolation of the phage SP6 RNA polymerase mutants. It was developed to isolate mutants with increased proficiency of termination at the SP6 terminator and/or with reduced elongation processivity. Mutations were randomly introduced into an N-terminal third of the polymerase gene that was placed under a lac promoter in one plasmid. In the other plasmid, a promoter-lacking lacZ gene modified for reduced translation efficiency was placed downstream of a tandem pair of the SP6 terminator located downstream of an SP6 promoter-chloramphenicol acetyltransferase gene. Termination-up mutants were selected in vivo as they rendered LacZ activity level lower than the wild-type, without reducing chloramphenicol resistance substantially. Three such mutants (M15L, M15S, and D117G) were purified, and their termination efficiencies were measured in vitro at two different intrinsic termination signals in the E. coli rrnB terminator t1 that are different in requiring RNA hairpin formation. All three mutations enhanced termination efficiencies in vitro at the SP6 terminator and the upstream signal of rrnB t1, but reduced the efficiency at the downstream signal of it. All the mutations reduced elongation processivity, as the mutants produced much less amounts of large transcripts (2.1 kb) than the wild-type but the similar amounts of small transcripts (up to 670 nt). Thus, the mutations, all reducing elongation processivity of the polymerase, exhibited opposite effects on the two types of intrinsic termination signals, suggesting that the two mechanisms involve different interactions with the phage RNA polymerase.
Yoo
J
J
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon, South Korea.
Kang
C
C
D019943:Amino Acid Substitution; D001435:Bacteriophages / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D005809:Genes, Regulator; D005822:Genetic Vectors; D007763:Lac Operon / Q000235:genetics; D009154:Mutation; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012323:RNA Processing, Post-Transcriptional; D012329:RNA, Bacterial / Q000235:genetics; D013728:Terminator Regions, Genetic
D017931:DNA Primers; D012329:RNA, Bacterial; C070852:RNA polymerase SP6; D012321:DNA-Directed RNA Polymerases