Structure-function analysis of human Spt4: evidence that hSpt4 and hSpt5 exert their roles in transcriptional elongation as parts of the DSIF complex.
BACKGROUND
The human Spt4/Spt5 complex, termed DRB-sensitivity inducing factor (DSIF) is a dual regulator of transcription that stimulates, or, when cooperating with negative elongation factor (NELF), represses RNA polymerase II (RNAPII) elongation. Spt4 and Spt5 are also thought to be involved in mRNA capping, homologous DNA recombination, and transcription-coupled DNA repair. As a first step to understanding how these proteins regulate diverse cellular processes, we investigated the structure and function of hSpt4 in vitro.


RESULTS
Immunodepletion of hSpt5 from HeLa nuclear extracts resulted in the efficient co-depletion of hSpt4. Using DSIF-depleted nuclear extracts and a series of Spt4 mutants, we examined the amino acid sequence of hSpt4 which was important for hSpt5 binding and for transcriptional repression and activation by DSIF. Unexpectedly, the zinc finger of hSpt4, which is critical for the yeast counterpart to function in vivo, was dispensable for hSpt5 binding and for transcriptional regulation in vitro.


CONCLUSIONS
These and other results suggest: (i) that the central region of hSpt4 is necessary and sufficient for its function in vitro and (ii) that there is no free hSpt4 or hSpt5 in cells. We propose that hSpt4 and hSpt5 exert their roles in transcriptional regulation, and possibly in other nuclear processes, as parts of the DSIF complex.
Kim
Dong-Ki
DK
Graduate School of Bioscience and Biotechnology, and Frontier Collaborative Research Center, Tokyo Institute of Technology, Yokohama 226-8501, Japan.
Inukai
Naoto
N
Yamada
Tomoko
T
Furuya
Akiko
A
Sato
Hiroe
H
Yamaguchi
Yuki
Y
Wada
Tadashi
T
Handa
Hiroshi
H
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D002478:Cells, Cultured; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry / Q000378:metabolism*; D015536:Down-Regulation; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D011994:Recombinant Proteins / Q000378:metabolism*; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D014158:Transcription, Genetic / Q000502:physiology; D035602:Transcriptional Elongation Factors / Q000737:chemistry / Q000378:metabolism*; D016335:Zinc Fingers / Q000502:physiology
D000911:Antibodies, Monoclonal; D002868:Chromosomal Proteins, Non-Histone; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; C481011:SUPT4H1 protein, human; C112224:SUPT5H protein, human; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; C118437:negative elongation factor; C069940:SPT5 transcriptional elongation factor