Evidence that the Tfg1/Tfg2 dimer interface of TFIIF lies near the active center of the RNA polymerase II initiation complex.
The ssu71 alleles of the TFG1 gene, which encodes the largest subunit of TFIIF, were isolated as suppressors of a TFIIB defect that affects the accuracy of transcription start site selection in the yeast Saccharomyces cerevisiae. Here we report that ssu71-1 also suppresses the cell growth and start site defects associated with an altered form of the Rpb1 subunit of RNA polymerase II (RNAP II). The ssu71-1 and ssu71-2 alleles were cloned and found to encode single amino acid replacements of glycine-363, either glycine to aspartic acid (G363D) or glycine to arginine (G363R). Two other charged replacements, G363E and G363K, were constructed by site-directed mutagenesis and suppress both TFIIB E62K and Rpb1 N445S, whereas neither G363A nor G363P exhibited any effect. G363 is phylogenetically conserved and its counterpart in human TFIIF (RAP74 G112) is located within the RAP74/RAP30 dimerization domain. We propose that the TFIIF dimerization domain is located in proximity to the B-finger of TFIIB near the active center of RNAP II where the TFIIB-TFIIF-RNAP II interface plays a key role in start site selection.
Freire-Picos
M Angeles
MA
Departamento de Bioloxía Celular e Molecular, Area de Bioquímica e Bioloxía Molecular, Campus da Zapateira S/N 1071 A, Coruña, Spain.
Krishnamurthy
Shankarling
S
Sun
Zu-Wen
ZW
Hampsey
Michael
M
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D019281:Dimerization; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D012319:RNA Polymerase II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013489:Suppression, Genetic; D035581:Transcription Factor TFIIB / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019473:Transcription Factors, TFII / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D024363:Transcription Initiation Site
C073680:SUA7 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D035581:Transcription Factor TFIIB; D019473:Transcription Factors, TFII; D012319:RNA Polymerase II; C056604:transcription factor TFIIF