Sequence and factor requirements for faithful in vitro transcription of human 7SL DNA

We have analysed the transcription of a functional human 7SL gene by RNA polymerase III (RNAPIII) in S100 extracts in vitro. Accurate and efficient synthesis of 7S L RNA depends on the presence of (i) an upstream sequence and (ii) an internal promoter element located within the first 22 bp of the gene. These findings were substantiated by DNase I footprinting. Mutations of the internal promoter identified the doublet CG [nucleotide (nt) +15/+16] outside the A-box homologue (nt +5 to +14) as being essential for both proper promoter function in the in vitro transcription assay and competition in the template-exclusion assay. Fractionation of S100 extracts identified two fractions required in addition to RNAPIII for faithful transcription of the gene. Each of these two fractions gave rise to one of two footprints observed in DNase I protection experiments, indicating that at least two DNA-binding factors are involved.