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 te rminator with bidirectional activity was localized between the transla tional termination codons of the streptokinase gene, skc, and the rel- orf1 genes. These two transcription units are oriented towards each ot her, and under normal conditions the site mRNA level exceeds that of t he rel-orf1 genes by a factor of at least 1000. Reporter vectors based on the promoterless cat gene were constructed by transcriptional fusi on of skc to cat, such that the region between the two genes contained the terminator in skc orientation or in rel-orf1 orientation. Additio nally, skc and cat were fused directly, with deletion of the terminato r. The reporter vectors were designed to be capable of being studied e ither as multicopy plasmids in Escherichia coli or in single copy foll owing integration, via skc, into the S. equisimilis chromosome. Chlora mphenicol acetyl transferase (CAT) activity assays in conjunction with determination of chloramphenicol resistance levels and Northern hybri dization analysis showed that the terminator is active in either host and orientation. However, termination efficiency was host dependent, w ith high terminator strength being observed in the homologous streptoc occal background and appreciable readthrough occurring in E. coli. The extent of transcriptional readthrough was dependent upon terminator o rientation, with termination being more efficient in rel-orf1 polarity . The results suggest that, in S. equisimilis, transcription of both s kc and rel-orf1 is efficiently terminated by a common signal, and that these genes are largely protected from convergent transcription, whic h otherwise would seem to be particularly detrimental to the weakly ex pressed rel-orf1 genes.