Isolation, Sequencing, and Mapping of the Human Homologue of the Yeast Transcription Factor, SPT5

Chiang, Pei Wen; Fogel, Eric; Jackson, Cynthia L.; Lieuallen, Kim; Lennon, Greg; Qu, Xuanhui; Wang, Su Qing; Kurnit, David M.

doi:10.1006/geno.1996.0646

Cited by 19 publications

(10 citation statements)

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“…The p160 cDNA that we have cloned is almost identical to these sequences. However, the deduced amino acid sequences which we and Stachora et al (27) report are different from that reported by Chiang et al (26) over two large portions, probably because of sequencing errors.…”

Section: Resultscontrasting

confidence: 93%

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Structure and Function of the Human Transcription Elongation Factor DSIF

Yamaguchi¹,

Wada

Watanabe

et al. 1999

Journal of Biological Chemistry

127

155

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5,6-Dichloro-1-␤-D-ribofuranosylbenzimidazole (DRB)is a classic inhibitor of transcription elongation by RNA polymerase II (pol II). We have previously identified and purified a novel transcription elongation factor, termed DSIF (for DRB sensitivity-inducing factor), that makes transcription sensitive to DRB. DSIF is composed of 160-and 14-kDa subunits, which are homologs of the Saccharomyces cerevisiae transcription factors Spt5 and Spt4. DSIF may either repress or stimulate transcription in vitro, depending on conditions, but its physiological function remains elusive. Here we characterize the structure and function of DSIF p160. p160 is shown to be a ubiquitous nuclear protein that forms a stable complex with p14 and interacts directly with the pol II largest subunit. Mutation analysis of p160 is used to identify structural features essential for its in vitro activity and to map the domains required for its interaction with p14 and pol II. Finally, a p160 mutant that represses DSIF activity in a dominant-negative manner is identified and used to demonstrate that DSIF represses transcription from various promoters in vivo.The nucleoside analog DRB 1 is a classic inhibitor of transcription elongation by pol II (reviewed in Ref. 1). Although it has been used for more than three decades, its mode of action has long been a mystery. DRB is unique in that it shows no effect on transcription reconstituted with purified general transcription factors (GTFs) and pol II, whereas it potently represses transcription in cruder systems or in vivo (2-4). Therefore, one or more factors apart from GTFs and pol II appear to be involved in DRB-sensitive transcription (1). Such putative factors must play general roles in pol II transcription in vivo because DRB affects most of the class II genes (4 -6).Recently, we and others have identified two elongation factors essential for DRB-sensitive transcription. One of them, positive transcription elongation factor b (P-TEFb), is a protein kinase that phosphorylates the pol II C-terminal domain (CTD) in a DRB-sensitive fashion (7-12). The CTD phosphorylation likely plays a pivotal role in pol II elongation and may be relevant to the P-TEFb function. The other, DSIF, has been purified from HeLa cell nuclear extract, based on its ability to induce DRB-sensitivity in vitro (3). DSIF is composed of 160-and 14-kDa subunits, which are homologs of the Saccharomyces cerevisiae transcription factors Spt5 and Spt4 (3, 13-15). DSIF/Spt4-Spt5 genetically and physically interacts with pol II, and thus may directly regulate pol II processivity (3, 16).The function of DSIF in the absence of DRB remains obscure. Small amounts of DSIF, when added back to a DSIF-depleted transcription system, repress transcription only in the presence of DRB, without affecting transcription in its absence (3). Higher doses of DSIF, however, repress transcription even in the absence of DRB (3). In contrast, under limiting concentrations of NTPs, DSIF stimulates transcription elongation in the absence of DRB (3). Genetic a...

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Section: Resultscontrasting

confidence: 93%

“…Two groups have reported nucleotide sequences of SUPT5H, a human homolog of S. cerevisiae SPT5 (26,27). The p160 cDNA that we have cloned is almost identical to these sequences.…”

Section: Resultsmentioning

confidence: 75%

Structure and Function of the Human Transcription Elongation Factor DSIF

Yamaguchi¹,

Wada

Watanabe

et al. 1999

Journal of Biological Chemistry

127

155

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“…The CTR2 region is a candidate for such phosphorylation, as it is known that the C‐terminal domain in RNA polymerase II can be phosphorylated by several protein kinases [25]. Another human amino acid sequence homologous to Spt5p has been published recently [26]. It lacks the second repeat motiv and diverges considerably from the one presented here, that is corroborated by peptide sequences derived from the purified protein.…”

Section: Discussionsupporting

confidence: 64%

Human Supt5h protein, a putative modulator of chromatin structure, is reversibly phosphorylated in mitosis

et al. 1997

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The Saccharomyces cerevisiae proteins Spt4p, Spt5p and Spt6p are involved in transcriptional repression by modulating the structure of chromatin. From HeLa cells we have purified a human homologue of Spt5p, SuptShp, and show here that the protein is reversibly phosphorylated in mitosis. The cloned cDNA predicts a protein of 1087 residues with 31% identity to yeast Spt5p. It includes an acidic N-terminus, a putative nuclear localization signal and a C-terminal region containing two different repeated motifs. One of them, with the consensus sequence P-T/S-P-S-P-Q/A-S/G-Y, is similar to the C-terminal domain in the largest subunit of RNA polymerase II.

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“…5A; Chiang et al 1996a;Stachora et al 1997). DSIF p160 has two striking features: a very acidic amino terminus and a carboxy-terminal amino acid repeat.…”

Section: Dsif P160 Is a Homolog Of The Yeast Transcription Factor Spt5mentioning

confidence: 99%

DSIF, a novel transcription elongation factor that regulates RNA polymerase II processivity, is composed of human Spt4 and Spt5 homologs

Wada

Takagi

Yamaguchi

et al. 1998

Genes & Development

657

707

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We report the identification of a transcription elongation factor from HeLa cell nuclear extracts that causes pausing of RNA polymerase II (Pol II) in conjunction with the transcription inhibitor 5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole (DRB). This factor, termed DRB sensitivity-inducing factor (DSIF), is also required for transcription inhibition by H8. DSIF has been purified and is composed of 160-kD (p160) and 14-kD (p14) subunits. Isolation of a cDNA encoding DSIF p160 shows it to be a homolog of the Saccharomyces cerevisiae transcription factor Spt5. Recombinant Supt4h protein, the human homolog of yeast Spt4, is functionally equivalent to DSIF p14, indicating that DSIF is composed of the human homologs of Spt4 and Spt5. In addition to its negative role in elongation, DSIF is able to stimulate the rate of elongation by RNA Pol II in a reaction containing limiting concentrations of ribonucleoside triphosphates. A role for DSIF in transcription elongation is further supported by the fact that p160 has a region homologous to the bacterial elongation factor NusG. The combination of biochemical studies on DSIF and genetic analysis of Spt4 and Spt5 in yeast, also in this issue, indicates that DSIF associates with RNA Pol II and regulates its processivity in vitro and in vivo.

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Isolation, Sequencing, and Mapping of the Human Homologue of the Yeast Transcription Factor, SPT5

Cited by 19 publications

References 0 publications

Structure and Function of the Human Transcription Elongation Factor DSIF

Structure and Function of the Human Transcription Elongation Factor DSIF

Human Supt5h protein, a putative modulator of chromatin structure, is reversibly phosphorylated in mitosis

DSIF, a novel transcription elongation factor that regulates RNA polymerase II processivity, is composed of human Spt4 and Spt5 homologs

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