Unusual Nucleic Acid Binding Properties of Factor 2, an RNA Polymerase II Transcript Release Factor

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We obtained protein sequence information from Drosophila factor 2, an ATP-dependent RNA polymerase II transcription termination factor, and discovered that it was identical to a SWI2/SNF2 family member called lodestar. Portions of putative human and Caenorhabditis elegans homologues were found in the sequence data bases and a complete cDNA for the human factor was generated using polymerase chain reaction techniques. Recombinant human factor 2 was produced in a baculovirus expression system, purified, and characterized. Similar to the authentic Drosophila factor, the human factor displayed a strong double-stranded DNA-dependent ATPase activity that was inhibited by singlestranded DNA and exhibited RNA polymerase II termination activity. Both factors were able to work on elongation complexes from either species. We discuss the mechanism of termination by factor 2 and the implications for the role of factor 2 in cellular activities.

Section: Resultsmentioning

confidence: 99%

Section: Human Rna Polymerase II Termination Factor 25542mentioning

confidence: 99%

Section: Cloning Expression and Production Of Antibodies To Drosophilamentioning

confidence: 99%

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A Human RNA Polymerase II Transcription Termination Factor Is a SWI2/SNF2 Family Member

Liu¹,

Zhi²,

Price³

1998

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“…TTF2 is a transcription termination factor and its dsDNA-dependent ATPase activity is required for releasing transcripts from the template (39). In the presence of ATP, TTF2 preferentially binds with ssDNA, and its ATPase activity is thus suppressed (40). To limit the impact of contaminating TTF2 during prephosphorylation reactions and subsequent transcription reactions, we carried out a "pre-termination" reaction to inhibit the continuous activity of TTF2.…”

Section: Methodsmentioning

confidence: 99%

Properties of RNA Polymerase II Elongation Complexes Before and After the P-TEFb-mediated Transition into Productive Elongation

Cheng

Price

2007

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138

178

The positive transcription elongation factor, P-TEFb, controls the fraction of initiated RNA polymerase II molecules that enter into the productive mode of elongation necessary to generate mRNAs. To better understand the mechanism of this transition into productive elongation we optimized a defined in vitro transcription system and compared results obtained with it to those obtained with a crude system. We found that controlling the function of TFIIF is a key aspect of RNA polymerase II elongation control. Before P-TEFb function, early elongation complexes under the control of negative factors are completely unresponsive to the robust elongation stimulatory activity of TFIIF. P-TEFb-mediated phosphorylation events, targeting the elongation complex containing DSIF and NELF, reverse the negative effect of DSIF and NELF and simultaneously facilitate the action of TFIIF. We also found that productive elongation complexes are completely resistant to negative elongation factors. Our data suggest that an additional factor(s) is involved in establishing the unique resistance activities of the elongation complexes before and after P-TEFb function. Furthermore, we provide evidence for the existence of another positive activity required for efficient function of P-TEFb. A model of the mechanism of P-TEFb-mediated elongation control is proposed in which P-TEFb induces the transition into productive elongation by changing the accessibility of elongation factors to elongation complexes. Our results have uncovered important properties of elongation complexes that allow a more complete understanding of how P-TEFb controls the elongation phases of transcription by RNA polymerase II.The elongation stage of eukaryotic RNA polymerase II (RNAPII) 2 transcription is not only essential for generating fulllength mRNA but also is a critical target for the regulation of gene expression (1, 2). An elongation control process was initially uncovered during studies of the transcription inhibitory mechanism of the ATP analog DRB (3, 4). DRB treatment blocks RNAPII transcription specifically at an early step in elongation without inhibiting the enzymatic activity of purified RNAPII itself (4, 5). Further studies uncovered a new class of elongation factors responsible for this DRB sensitivity. Two negative elongation factors, the DRB sensitivity-inducing factor (DSIF) and the negative elongation factor (NELF) cause transcription pausing by physically associating with RNAPII (6 -8). Positive transcription elongation factor b (P-TEFb), a cyclindependent kinase that can be inhibited by DRB, counteracts the negative effects of DSIF and NELF and allows RNAPII to enter productive elongation (9 -14).Early studies identified many genes that were regulated at the stage of transcription elongation, including hsp70 (15), c-myc (16,17), and the HIV-LTR (18), and later a common regulatory mechanism utilized by these genes was uncovered, which was the recruitment of P-TEFb to the transcription machinery (12,19,20). Indeed, P-TEFb is the key regulator that ca...

“…Support for this view comes from the recent identification of the negative elongation factors 5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole (DRB) sensitivityinducing factor, negative elongation factor, and factor 2 and the positive elongation factor P-TEFb. Factor 2 is an ATP-dependent termination factor that releases transcripts associated with stalled RNAPII molecules (2). DSIF is a repressor of elongation that was identified as a factor that renders in vitro transcription reactions sensitive to the drug DRB (3).…”

mentioning

confidence: 99%

Cyclin K Functions as a CDK9 Regulatory Subunit and Participates in RNA Polymerase II Transcription

Fu¹,

Peng²,

Lee³

et al. 1999

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205

181

Important progress in the understanding of elongation control by RNA polymerase II (RNAPII) has come from the recent identification of the positive transcription elongation factor b (P-TEFb) and the demonstration that this factor is a protein kinase that phosphorylates the carboxyl-terminal domain (CTD) of the RNAPII largest subunit. The P-TEFb complex isolated from mammalian cells contains a catalytic subunit (CDK9), a cyclin subunit (cyclin T1 or cyclin T2), and additional, yet unidentified, polypeptides of unknown function. To identify additional factors involved in P-TEFb function we performed a yeast two-hybrid screen using CDK9 as bait and found that cyclin K interacts with CDK9 in vivo. Biochemical analyses indicate that cyclin K functions as a regulatory subunit of CDK9. The CDK9-cyclin K complex phosphorylated the CTD of RNAPII and functionally substituted for P-TEFb comprised of CDK9 and cyclin T in in vitro transcription reactions.Accumulating evidence indicates that the expression of many protein coding genes is regulated at the level of transcription elongation. Understanding of elongation control by RNAPII has been hampered by slow progress in the identification of factors involved in transcription elongation. An emerging model is that the interplay of positive and negative elongation factors determines the elongation potential of RNAPII 1 in different promoters (1). Support for this view comes from the recent identification of the negative elongation factors 5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole (DRB) sensitivityinducing factor, negative elongation factor, and factor 2 and the positive elongation factor P-TEFb. Factor 2 is an ATP-dependent termination factor that releases transcripts associated with stalled RNAPII molecules (2). DSIF is a repressor of elongation that was identified as a factor that renders in vitro transcription reactions sensitive to the drug DRB (3). NELF works in conjunction with DSIF to repress RNA polymerase II elongation (4). P-TEFb is a DRB-sensitive kinase that is believed to stimulate the elongation potential of RNAPII by phosphorylating the CTD of RNAPII molecules that are engaged in early transcription elongation (5, 6). It was recently suggested that P-TEFb-mediated phosphorylation of the CTD prevents the association of DSIF with RNAPII and thereby overcomes DSIFdependent repression (7). Although it has been long accepted that CTD phosphorylation plays a critical role in transcription, it has been difficult to ascertain the mammalian kinases responsible for CTD phosphorylation in vivo. The observation that the ability of several drugs to block CTD phosphorylation in vivo correlates with the ability of these compounds to inhibit P-TEFb in vitro strongly suggests that P-TEFb might indeed function as a CTD kinase in vivo (8). Additional evidence showing that PTEFb kinase functions as a positive elongation factor in vivo comes from studies with the HIV Tat protein that have shown that the catalytic activity of P-TEFb is required for Tat-dependent stimulation of tra...