Role of a small RNA pol II subunit in TATA to transcription start site spacing.

The yeast shi mutation affects the spacing between the TATA promoter element and transcription initiation sites; for the H2B and ADH1 genes, a series of start sites located approximately 50-80 bp downstream of TATA is used in addition to the wild-type initiation sites located at around 100 bp from TATA (1). Here, the yeast SHI wild-type gene has been isolated by complementation and shown to be identical to RPB9, the gene encoding a small subunit of RNA polymerase II. A point mutation in the shi gene, changing a cysteine residue in a putative zinc ribbon motif into a phenylalanine residue, was demonstrated to permit the observed usage of upstream initiation sites. Deletion of the non-essential SHI gene also results in usage of upstream initiation sites and causes conditional growth defects

Promoter-Specific Shifts in Transcription Initiation Conferred by Yeast TFIIB Mutations Are Determined by the Sequence in the Immediate Vicinity of the Start Sites

The general transcription factor IIB (TFIIB) is required for transcription of class II genes by RNA polymerase II. Previous studies demonstrated that mutations in the Saccharomyces cerevisiae SUA7 gene, which encodes TFIIB, can alter transcription initiation patterns in vivo. To further delineate the functional domain and residues of TFIIB involved in transcription start site utilization, a genetic selection was used to isolate S. cerevisiae TFIIB mutants exhibiting downstream shifts in transcription initiation in vivo. Both dominant and recessive mutations conferring downstream shifts were identified at multiple positions within a highly conserved homology block in the N-terminal region of the protein. The TFIIB mutations conferred downstream shifts in transcription initiation at the ADH1 and CYC1 promoters, whereas no significant shifts were observed at the HIS3 promoter. Analysis of a series of ADH1-HIS3 hybrid promoters and variant ADH1 and HIS3 promoters containing insertions, deletions, or site-directed base substitutions revealed that the feature that renders a promoter sensitive to TFIIB mutations is the sequence in the immediate vicinity of the normal start sites. We discuss these results in light of possible models for the mechanism of start site utilization by S. cerevisiae RNA polymerase II and the role played by TFIIB