Structure–function analysis of human Spt4: evidence that hSpt4 and hSpt5 exert their roles in transcriptional elongation as parts of the DSIF complex
✍ Scribed by Dong-ki Kim; Naoto Inukai; Tomoko Yamada; Akiko Furuya; Hiroe Sato; Yuki Yamaguchi; Tadashi Wada; Hiroshi Handa
- Book ID
- 104460478
- Publisher
- John Wiley and Sons
- Year
- 2003
- Tongue
- English
- Weight
- 262 KB
- Volume
- 8
- Category
- Article
- ISSN
- 1356-9597
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✦ Synopsis
Abstract
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.
Conclusion: 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.