[Show abstract][Hide abstract] ABSTRACT: Hepatitis delta virus (HDV) is an RNA virus whose replication and transcription are considered to proceed via RNA-dependent RNA synthesis by RNA polymerase II (Pol II), and the viral protein called hepatitis delta antigen (HDAg) is essential for these processes. HDAg was previously shown to stimulate Pol II elongation on both DNA and RNA templates in vitro. Here, the mechanism of elongation control by HDAg was investigated because it serves as a prototype of cellular transcription elongation factors and also plays an interesting role in HDV proliferation. With site-specific photocrosslinking and transcription using reconstituted elongation complexes, evidence is presented that HDAg functionally interacts with the clamp of Pol II, a mobile structure that holds DNA and RNA in place. Strikingly, HDAg not only increases the rate of elongation but also affects the decision of which nucleotide is incorporated. These and our previous findings lead us to propose a model in which HDAg interacts with and loosens the clamp, and thereby accelerates forward translocation of Pol II at the cost of fidelity. By reducing transcriptional fidelity in terms of not only discrimination of incoming nucleotides but also recognition of templates, HDAg may facilitate the unusual RNA-dependent RNA synthesis by Pol II.
[Show abstract][Hide abstract] ABSTRACT: Human DSIF, a heterodimer composed of hSpt4 and hSpt5, plays opposing roles in transcription elongation by RNA polymerase II (RNA Pol II). Here, we describe an evolutionarily conserved repetitive heptapeptide motif (consensus = G-S-R/Q-T-P) in the C-terminal region (CTR) of hSpt5, which, like the C-terminal domain (CTD) of RNA Pol II, is highly phosphorylated by P-TEFb. Thr-4 residues of the CTR repeats are functionally important phosphorylation sites. In vitro, Thr-4 phosphorylation is critical for the elongation activation activity of DSIF, but not to its elongation repression activity. In vivo, Thr-4 phosphorylation is critical for epidermal growth factor (EGF)-inducible transcription of c-fos and for efficient progression of RNA Pol II along the gene. We consider this phosphorylation to be a switch that converts DSIF from a repressor to an activator. We propose the "mini-CTD" hypothesis, in which phosphorylated CTR is thought to function in a manner analogous to phosphorylated CTD, serving as an additional code for active elongation complexes.