Primary structure requirements for in vivo activity and bidirectional function of the transcription terminator shared by the oppositely oriented skc/rel-orf1 genes of Streptococcus equisimilis H46A.
The region between the Streptococcus equisimilis streptokinase (skc) gene and the oppositely oriented rel-orf1 transcription unit contains only one termination site known to function bidirectionally in both the homologous host and in Escherichia coli. The terminator sequence is similar to other factor-independent terminators. Using two sets of point mutations that interrupt the hairpin-upstream oligo(dA) tract or the hairpin-downstream oligo(dT) tract, we examined the possible contribution of extended base pairing between the upstream rA and downstream rU residues to efficient termination and bidirectionality in both hosts, using terminator-cat reporter gene fusions in either polarity. The results show that interrupting the oligo(dA) tract preceding the hairpin has relatively little effect on terminator strength in either orientation in the homologous host, but abolishes termination in skc polarity in E. coli. Disruption of the hairpin-distal oligo(dT) tract inactivated the terminator in skc polarity in both hosts, had little effect on termination efficiency in rel-orf1 polarity in S. equisimilis, and also retained appreciable terminator activity in E. coli. In general, these alterations of the terminator sequence, together with additional mutations that reduce the spacing between the skc stop codon and the termination site or introduce a base substitution in the terminator stem, adversely affected the efficiency of termination to a greater extent in E. coli than in the homologous host. The disparity between the effects of certain mutations in the two hosts suggests that, in addition to thermodynamic properties, specific host factors, including RNA polymerase, contribute to terminator strength.
Steiner
K
K
Institute for Molecular Biology, Jena University, Germany.
Malke
H
H
D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D018388:Codon, Terminator / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D017930:Genes, Reporter; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics; D013291:Streptococcus / Q000201:enzymology / Q000235:genetics*; D013300:Streptokinase / Q000235:genetics*; D013728:Terminator Regions, Genetic; D014158:Transcription, Genetic
D018388:Codon, Terminator; D017931:DNA Primers; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D015500:Chloramphenicol O-Acetyltransferase; D013300:Streptokinase