Characterization of a HeLa cDNA clone encoding the human SII protein, an elongation factor for RNA polymerase II

Chen, H.Carrie; England, Laura; Kane, Caroline M.

doi:10.1016/0378-1119(92)90522-q

Cited by 26 publications

(16 citation statements)

References 19 publications

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“…Predicted amino acid sequences of Elongin A3 revealed several notable structural features. First, like Elongins A and A2, the NH 2 -terminal ϳ110 amino acids of Elongin A3 resembled the NH 2 terminus of transcription elongation factor SII (29% identity to human SII) (9,10,22). Second, Elongin A3 also possesses the Elongin BC binding sequence (residues 336 -347) at its COOH terminus.…”

Section: Resultsmentioning

confidence: 99%

Identification and Biochemical Characterization of a Novel Transcription Elongation Factor, Elongin A3

Yamazaki¹,

Guo²,

Sugahara³

et al. 2002

Journal of Biological Chemistry

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The Elongin complex stimulates the rate of transcription elongation by RNA polymerase II by suppressing the transient pausing of the polymerase at many sites along the DNA template. Elongin is composed of a transcriptionally active A subunit and two small regulatory B and C subunits, the latter binding stably to each other to form a binary complex that interacts with Elongin A and strongly induces its transcriptional activity. To further understand the role of Elongin A in transcriptional regulation by RNA polymerase II, we are attempting to identify Elongin A-related proteins. Here, we report on the molecular cloning, expression, and biochemical characterization of human Elongin A3, a novel transcription elongation factor that exhibits 49 and 81% identity to Elongin A and the recently identified Elongin A2, respectively. The mRNA of Elongin A3 is ubiquitously expressed, and the protein is localized to the nucleus of cells. Mechanistic studies have demonstrated that Elongin A3 possesses similar biochemical features to Elongin A2. Both stimulate the rate of transcription elongation by RNA polymerase II and are capable of forming a stable complex with Elongin BC. In contrast to Elongin A, however, their transcriptional activities are not activated by Elongin BC. Structure-function analyses using fusion proteins composed of Elongin A3 and Elongin A revealed that the COOH-terminal region of Elongin A is important for the activation by Elongin BC.The synthesis of messenger RNA in eukaryotes is a complex biochemical process controlled by the concerted action of a set of general transcription factors that regulate the activity of RNA polymerase II during the initiation and elongation stages of transcription. At least six general transcription initiation factors (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH) 1 have been identified in eukaryotic cells and found to promote the selective binding of RNA polymerase II to promoters and to support a basal level of transcription (1). In addition to the general initiation factors, at least 16 elongation factors have been defined biochemically and found to increase the efficiency of transcription elongation by RNA polymerase II (2-5).Among the elongation factors, SII and P-TEFb prevent RNA polymerase II from prematurely arresting transcription. SII does this by promoting passage of the polymerase through a variety of transcriptional impediments, including DNA sequences that act as intrinsic arrest sites and DNA-bound proteins and drugs (6). P-TEFb catalyzes the conversion of early, termination-prone elongation complexes into productive elongation complexes (7). The rest of the elongation factors such as TFIIF (8), Elongin A (9), Elongin A2 (10), ELL (11), and the Cockayne syndrome group B protein (CSB) (12), act to increase the overall rate of RNA chain elongation by RNA polymerase II by decreasing the frequency and/or duration of transient pausing of the polymerase at sites along the DNA template.Elongin was initially identified as a heterotrimer composed of A, B, and C subunits...

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Section: Resultsmentioning

confidence: 99%

Identification and Biochemical Characterization of a Novel Transcription Elongation Factor, Elongin A3

Yamazaki¹,

Guo²,

Sugahara³

et al. 2002

Journal of Biological Chemistry

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“…The cDNA of the HeLa elongation factor TFIIS predicts a polypeptide of 301 amino acids (24). A deletion mutant expressing the last 171 amino acids still exhibits elongation stimulatory activity when assayed on either salmon sperm DNA (data not shown) or 3' extended DNA templates (described below).…”

Section: Resultsmentioning

confidence: 99%

“…A comparison of the protein sequences predicted from the clones for the various TFIIS related proteins reveals striking sequence similarities, but the conservation is especially prominent in the C-terminal regions (24). Therefore, the C-terminal 171 amino acids of the HeLa cDNA (ATFIIS)…”

Section: Resultsmentioning

confidence: 99%

Cleavage of the nascent transcript induced by TFIIS is insufficient to promote read-through of intrinsic blocks to elongation by RNA polymerase II.

Cipres-Palacin

Kane

1994

Proc. Natl. Acad. Sci. U.S.A.

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RNA polymerases encounter a variety of types of blocks to elongation during transcription in eukaryotic cells. At least one protein, THIS, can promote read-through of many types of blocks to elongation by RNA polymerase II, and this protein stimulates cleavage of the nascent transcript in stalled elongation complexes as a prelude to read-through. The C-terminal halfofthe TFIIS protein is sufficient for stimulating the cleavage and read-through reactions in vitro. To Recently it has been suggested that TFIIS may operate in part by inducing cleavage of the 3' end of the nascent transcript in the ternary elongation complex (14,16,18). This cleavage reaction allows the polymerase to read through a template region that is otherwise only partially permissive for elongation (15). In a similar functional reaction, prokaryotic factors GreA and GreB stimulate cleavage in association with Escherichia coli RNA polymerase (19,20).At any block to elongation, only a fraction of the elongating polymerases stops transcription while the remainder reads through the block even in the absence of added factors (7,8,15). For those stalled polymerases, the transcript cleavage event stimulated by TFIIS precedes read-through (15). Following cleavage, read-through itself was thought to involve the passive resynthesis of RNA by RNA polymerase. Since only a fraction of polymerases would read through the block in any one cycle, repetitive cycles of cleavage and resynthesis would be necessary for all RNA polymerases eventually to read through the block (21). This hypothesis would predict that promoting the cleavage reaction would be the primary if not the only function of TFIIS in such read-through reactions. However, the results described here suggest that the read-through mechanism involving TFIIS cannot be explained so simply, as mutant TFIIS proteins, which are essentially inactive for promoting read-through, nonetheless stimulate efficient cleavage in stalled ternary elongation complexes.EXPERIMENTAL PROCEDURES Materials. Inhibit-ACE was obtained from 5'--3', Inc.Nucleotides were obtained from Pharmacia. [a-32P]CTP (>400 Ci/mmol; 1 Ci = 37 GBq) was obtained from Amersham. Bio-Gel 30 columns were obtained from Bio-Rad. His-Bind metal chelation resin was from Novagen. RNA polymerase II was purified from calf thymus as described (22).Strains and Plasmids. E. coli strain CJ236 (durt, ung-) was used to prepare uracil-containing template DNA for mutagenesis (23). A fragment containing the last 171 amino acids of the open reading frame of HeLa TFIIS (24), fused to 6 histidines at its N terminus, was prepared using the polymerase chain reaction (PCR) (25). The PCR product was digested with Nde I and BamHI and then ligated into the 5.4-kb Nde I-BamHI-digested pET22b(+) (Novagen). This plasmid is referred to as pET22b(+)/ATFIIS, and the protein produced from this plasmid is referred to as ATFIIS.Production ofMutants. Two synthetic oligonucleotides were constructed to mutate the charged amino acids E173, E174 (creating TFIIS5) or K186, K1...

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“…Z54216), and fission and baker's yeast (Nakanishi et al, 1992;Williams and Kane, 1996;Marshall et al, 1990;Plant et al, 1996;Kugawa et al, 1996;Labhart, 1997a;Hirashima et al, 1988;Xu et al, 1994;Chen et al, 1992), as well as from archaebacteria and viruses (Langer and Zillig, 1993;Yamada et al, 1992;Rodriguez et al, 1992). More distantly related proteins include the 30-kDa subunit of vaccinia virus RNA polymerase (Ahn et al, 1990), a 21-kDa protein encoded by a HeLa cell cDNA (Yeh and Shatkin, 1994), and elongin A (Aso et al, 1995).…”

Section: Introductionmentioning

confidence: 96%

Identification of Novel Genes Encoding Transcription Elongation Factor TFIIS (TCEA) in Vertebrates: Conservation of Three Distinct TFIIS Isoforms in Frog, Mouse, and Human

Labhart

Morgan

1998

Genomics

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Characterization of a HeLa cDNA clone encoding the human SII protein, an elongation factor for RNA polymerase II

Cited by 26 publications

References 19 publications

Identification and Biochemical Characterization of a Novel Transcription Elongation Factor, Elongin A3

Identification and Biochemical Characterization of a Novel Transcription Elongation Factor, Elongin A3

Cleavage of the nascent transcript induced by TFIIS is insufficient to promote read-through of intrinsic blocks to elongation by RNA polymerase II.

Identification of Novel Genes Encoding Transcription Elongation Factor TFIIS (TCEA) in Vertebrates: Conservation of Three Distinct TFIIS Isoforms in Frog, Mouse, and Human

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