Interaction of yeast transcription factor IIIC with dimeric Schizosaccharomyces pombe tRNA(Ser)-tRNA(Met) genes.
A unique tRNA(Ser)-tRNA(Met) tandem gene arrangement was characterized previously from Schizosaccharomyces pombe. Three alleles exist in which a tRNA(Ser) gene is separated by 7 base pairs from an initiator tRNA(Met) gene. Promotion of transcription occurs only within the tRNA(Ser) gene, yielding a dimeric precursor transcript. Using nuclease protection and gel retardation assays, we have analyzed how the Saccharomyces cerevisiae RNA polymerase III transcription factor C (TFIIIC) interacts with this dimeric gene template. The primary interaction site of TFIIIC with the tRNA(Ser) gene is at the 3'-internal control region (ICR), which can be distinguished kinetically from its weaker interaction with the 5'-ICR of the gene. We examined a variety of point mutations and double mutations within the tRNA(Ser) gene which reduce transcription. We found that changes in highly conserved nucleotides within the ICRs reduce TFIIIC binding up to 7-fold compared with the parent suppressor gene. The interaction of TFIIIC with the tRNA(Ser) gene does not sterically prevent stable binding of TFIIIC to the 3'-ICR of the tRNA(Met) gene. However, the affinity of binding of TFIIIC to the dimeric template is 7-fold higher than to the tRNA(Met) gene, alone, demonstrating that the tRNA(Met) gene contains intrinsically weak promoter elements. This may contribute to the inability of the tRNA(Met) gene to independently direct transcription from its ICR elements.
Johnson
D L
DL
School of Pharmacy, Division of Biomedicinal Chemistry, University of Southern California, Los Angeles 90033.
Nichols
M
M
Bolger
M B
MB
Wilson
S
S
D000483:Alleles; D001483:Base Sequence; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D012320:RNA Polymerase III / Q000378:metabolism; D012345:RNA, Transfer, Amino Acid-Specific / Q000235:genetics*; D012358:RNA, Transfer, Met / Q000235:genetics*; D012362:RNA, Transfer, Ser / Q000235:genetics*; D004718:Saccharomycetales / Q000235:genetics*; D012568:Schizosaccharomyces / Q000235:genetics* / Q000378:metabolism; D013698:Templates, Genetic; D014157:Transcription Factors / Q000378:metabolism*; D019474:Transcription Factors, TFIII; D014158:Transcription, Genetic; D000098832:Transcription Factor TFIIIC
D012345:RNA, Transfer, Amino Acid-Specific; D012358:RNA, Transfer, Met; D012362:RNA, Transfer, Ser; D014157:Transcription Factors; D019474:Transcription Factors, TFIII; D000098832:Transcription Factor TFIIIC; D012320:RNA Polymerase III