Intragenic promoter adaptation and facilitated RNA polymerase III recycling in the transcription of SCR1, the 7SL RNA gene of Saccharomyces cerevisiae.
The SCR1 gene, coding for the 7SL RNA of the signal recognition particle, is the last known class III gene of Saccharomyces cerevisiae that remains to be characterized with respect to its mode of transcription and promoter organization. We show here that SCR1 represents 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 TpsiC 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, SCR1 transcription 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 vitro and 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 vivo than in vitro transcription. Transcriptional competition and nucleosome disruption experiments showed that this stronger in vivo defect is due to a reduced ability of A-block-mutated SCR1 to 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 SCR1 gene, the longest known class III transcriptional unit.
Dieci
Giorgio
G
Dipartimento di Biochimica e Biologia Molecolare, Università di Parma, I-43100 Parma, Italy. gdieci@unipr.it
Giuliodori
Silvia
S
Catellani
Manuela
M
Percudani
Riccardo
R
Ottonello
Simone
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002843:Chromatin / Q000737:chemistry / Q000378:metabolism; D003001:Cloning, Molecular; D003850:Deoxyribonuclease I / Q000378:metabolism; D007700:Kinetics; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009707:Nucleosomes / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012313:RNA / Q000378:metabolism; D012320:RNA Polymerase III / Q000235:genetics*; D020733:RNA, Small Cytoplasmic / Q000378:metabolism*; D012343:RNA, Transfer / Q000378:metabolism; D011951:Receptors, Complement / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000378:metabolism; D018271:Signal Recognition Particle / Q000378:metabolism*; D019474:Transcription Factors, TFIII / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D000098832:Transcription Factor TFIIIC
C115990:7SL RNA; D002843:Chromatin; D009707:Nucleosomes; D020733:RNA, Small Cytoplasmic; D011951:Receptors, Complement; D018271:Signal Recognition Particle; D019474:Transcription Factors, TFIII; D000098832:Transcription Factor TFIIIC; C082885:soluble complement inhibitor 1; D012313:RNA; D012343:RNA, Transfer; D012320:RNA Polymerase III; D003850:Deoxyribonuclease I