Transcription termination of the streptokinase gene of Streptococcus equisimilis H46A: bidirectionality and efficiency in homologous and heterologous hosts.
In Streptococcus equisimilis H46A, a hypersymmetrical transcription terminator with bidirectional activity was localized between the translational termination codons of the streptokinase gene, skc, and the rel-orf1 genes. These two transcription units are oriented towards each other, and under normal conditions the skc mRNA level exceeds that of the rel-orf1 genes by a factor of at least 1000. Reporter vectors based on the promoterless cat gene were constructed by transcriptional fusion of skc to cat, such that the region between the two genes contained the terminator in skc orientation or in rel-orf1 orientation. Additionally, skc and cat were fused directly, with deletion of the terminator. The reporter vectors were designed to be capable of being studied either as multicopy plasmids in Escherichia coli or in single copy following integration, via skc, into the S. equisimilis chromosome. Chloramphenicol acetyl transferase (CAT) activity assays in conjunction with determination of chloramphenicol resistance levels and Northern hybridization analysis showed that the terminator is active in either host and orientation. However, termination efficiency was host dependent, with high terminator strength being observed in the homologous streptococcal background and appreciable readthrough occurring in E. coli. The extent of transcriptional readthrough was dependent upon terminator orientation, with termination being more efficient in rel-orf1 polarity. The results suggest that, in S. equisimilis, transcription of both skc and rel-orf1 is efficiently terminated by a common signal, and that these genes are largely protected from convergent transcription, which otherwise would seem to be particularly detrimental to the weakly expressed rel-orf1 genes.
Steiner
K
K
Institute for Molecular Biology, Jena University, Germany.
Malke
H
H
D001483:Base Sequence; D018388:Codon, Terminator / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012329:RNA, Bacterial / Q000096:biosynthesis / Q000737:chemistry; D012333:RNA, Messenger / Q000096:biosynthesis / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013045:Species Specificity; D013291:Streptococcus / Q000201:enzymology / Q000235:genetics*; D013300:Streptokinase / Q000235:genetics*; D013728:Terminator Regions, Genetic / Q000235:genetics*; D014158:Transcription, Genetic / Q000235:genetics
D018388:Codon, Terminator; D012329:RNA, Bacterial; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D013300:Streptokinase