The highly conserved glutamic acid 791 of Rpb2 is involved in the binding of NTP and Mg(B) in the active center of human RNA polymerase II.

During transcription by RNA polymerase (RNAP) II, the incoming ribonucleoside triphosphate (NTP) enters the catalytic center in association with an Mg2+ ion, termed metal B [Mg(B)]. When bound to RNAP II, Mg(B) is coordinated by the beta and gamma phosphates of the NTP, Rpb1 residues D481 and D483 and Rpb2 residue D837. Rpb2 residue D837 is highly conserved across species. Notably, its neighboring residue, E836 (E791 in human RNAP II), is also highly conserved. To probe the role of E791 in transcription, we have affinity purified and characterized a human RNAP II mutant in which this residue was substituted for alanine. Our results indicate that the transcription activity of the Rpb2 E791A mutant is impaired at low NTP concentrations both in vitro and in vivo. They also revealed that both its NTP polymerization and transcript cleavage activities are decreased at low Mg concentrations. Because Rpb2 residue E791 appears to be located too far from the NTP-Mg(B) complex to make direct contact at either the entry (E) or addition (A) site, we propose alternative mechanisms by which this highly conserved residue participates in loading NTP-Mg(B) in the active site during transcription.

RPAP1, a novel human RNA polymerase II-associated protein affinity purified with recombinant wild-type and mutated polymerase subunits.

We have programmed human cells to express physiological levels of recombinant RNA polymerase II (RNAPII) subunits carrying tandem affinity purification (TAP) tags. Double-affinity chromatography allowed for the simple and efficient isolation of a complex containing all 12 RNAPII subunits, the general transcription factors TFIIB and TFIIF, the RNAPII phosphatase Fcp1, and a novel 153-kDa polypeptide of unknown function that we named RNAPII-associated protein 1 (RPAP1). The TAP-tagged RNAPII complex is functionally active both in vitro and in vivo. A role for RPAP1 in RNAPII transcription was established by shutting off the synthesis of Ydr527wp, a Saccharomyces cerevisiae protein homologous to RPAP1, and demonstrating that changes in global gene expression were similar to those caused by the loss of the yeast RNAPII subunit Rpb11. We also used TAP-tagged Rpb2 with mutations in fork loop 1 and switch 3, two structural elements located strategically within the active center, to start addressing the roles of these elements in the interaction of the enzyme with the template DNA during the transcription reaction.