| Abstrakt: |
The SCR1gene, coding for the 7SL RNA of the signal recognition particle, is the last known class III gene ofSaccharomyces cerevisiaethat remains to be characterized with respect to its mode of transcription and promoter organization. We show here that SCR1represents a unique case of a non-tRNA class III gene in which intragenic promoter elements (the TFIIIC-binding A- and B-blocks), corresponding to the D and TΨC arms of mature tRNAs, have been adapted to a structurally different small RNA without losing their transcriptional function. In fact, despite the presence of an upstream canonical TATA box, SCR1transcription strictly depends on the presence of functional, albeit quite unusual, A- and B-blocks and requires all the basal components of the RNA polymerase III transcription apparatus, including TFIIIC. Accordingly, TFIIIC was found to protect from DNase I digestion an 80-bp region comprising the A- and B-blocks. B-block inactivation completely compromised TFIIIC binding and transcription capacity in vitroand in vivo. An inactivating mutation in the A-block selectively affected TFIIIC binding to this promoter element but resulted in much more dramatic impairment of in vivothan in vitrotranscription. Transcriptional competition and nucleosome disruption experiments showed that this stronger in vivodefect is due to a reduced ability of A-block-mutated SCR1to compete with other genes for TFIIIC binding and to counteract the assembly of repressive chromatin structures through TFIIIC recruitment. A kinetic analysis further revealed that facilitated RNA polymerase III recycling, far from being restricted to typical small sized class III templates, also takes place on the 522-bp-long SCR1gene, the longest known class III transcriptional unit. |
