Studies of nematode TFIIE function reveal a link between Ser-5 phosphorylation of RNA polymerase II and the transition from transcription initiation to elongation.
The general transcription factor TFIIE plays important roles in transcription initiation and in the transition to elongation. However, little is known about its function during these steps. Here we demonstrate for the first time that TFIIH-mediated phosphorylation of RNA polymerase II (Pol II) is essential for the transition to elongation. This phosphorylation occurs at serine position 5 (Ser-5) of the carboxy-terminal domain (CTD) heptapeptide sequence of the largest subunit of Pol II. In a human in vitro transcription system with a supercoiled template, this process was studied using a human TFIIE (hTFIIE) homolog from Caenorhabditis elegans (ceTFIIEalpha and ceTFIIEbeta). ceTFIIEbeta could partially replace hTFIIEbeta, whereas ceTFIIEalpha could not replace hTFIIEalpha. We present the studies of TFIIE binding to general transcription factors and the effects of subunit substitution on CTD phosphorylation. As a result, ceTFIIEalpha did not bind tightly to hTFIIEbeta, and ceTFIIEbeta showed a similar profile for binding to its human counterpart and supported an intermediate level of CTD phosphorylation. Using antibodies against phosphorylated serine at either Ser-2 or Ser-5 of the CTD, we found that ceTFIIEbeta induced Ser-5 phosphorylation very little but induced Ser-2 phosphorylation normally, in contrast to wild-type hTFIIE, which induced phosphorylation at both Ser-2 and Ser-5. In transcription transition assays using a linear template, ceTFIIEbeta was markedly defective in its ability to support the transition to elongation. These observations provide evidence of TFIIE involvement in the transition and suggest that Ser-5 phosphorylation is essential for Pol II to be in the processive elongation form.
Yamamoto
S
S
Institute for Molecular and Cellular Biology, Osaka University, Suita, Osaka 565-0871, Japan.
Watanabe
Y
Y
van der Spek
P J
PJ
Watanabe
T
T
Fujimoto
H
H
Hanaoka
F
F
Ohkuma
Y
Y
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D017173:Caenorhabditis elegans / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D015801:Helminth Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012319:RNA Polymerase II / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D016415:Sequence Alignment; D013698:Templates, Genetic; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019473:Transcription Factors, TFII; D014158:Transcription, Genetic
D015801:Helminth Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D019473:Transcription Factors, TFII; C066644:transcription factor TFIIE; D010768:Phosphoserine; D012319:RNA Polymerase II