RNA polymerase II lacking the Rpb9 subunit uses alternate transcription initiation sites in vitro and in vivo and is unable to respond to the transcription elongation factor TFIIS in vitro. Here, we show that RPB9 has a synthetic phenotype with the TFIIS gene. Disruption of RPB9 in yeast also resulted in sensitivity to 6-azauracil, which is a phenotype linked to defects in transcription elongation. Expression of the TFIIS gene on a high-copy plasmid partially suppressed the 6-azauracil sensitivity of ⌬rpb9 cells. We set out to determine the relevant cellular role of yeast Rpb9 by assessing the ability of 20 different site-directed and deletion mutants of RPB9 to complement the initiation and elongation defects of ⌬rpb9 cells in vivo. Rpb9 is composed of two zinc ribbons. The N-terminal zinc ribbon restored the wild-type pattern of initiation start sites, but was unable to complement the growth defects associated with defects in elongation. Most of the site-directed mutants complemented the elongation-specific growth phenotypes and reconstituted the normal pattern of transcription initiation sites. The anti-correlation between the growth defects of cells disrupted for RPB9 and the selection of transcription start sites suggests that this is not the primary cellular role for Rpb9. Genome-wide transcription profiling of ⌬rpb9 cells revealed only a few changes, predominantly in genes related to metabolism.RNA polymerase II comprises 12 subunits in yeast (1). Four of the subunits, Rpb1, Rpb2, Rpb3, and Rpb11, form a catalytic core that is homologous in structure and function to the prokaryotic core RNA polymerase (2, 3). The other eight eukaryotic subunits are less well characterized. Five of these subunits, Rpb5, Rpb6, Rpb8, Rpb10, and Rpb12, are found in all three eukaryotic RNA polymerases (4 -6). The other three, Rpb4, Rpb7, and Rpb9, are unique to RNA polymerase II, although both Rpb7 and Rpb9 have sequence homologues in RNA polymerases I and III (7). The gene for Rpb9 is not essential for yeast cell viability, but is essential in Drosophila (8).Rpb9 has roles both in transcription initiation and in transcription elongation. In the initiation reaction, Rpb9 modulates the selection of the transcription start site. In cells lacking Rpb9 and in reconstituted transcription reactions lacking Rpb9, the population of start sites is shifted upstream at a variety of promoters (9 -11). In the elongation reaction, Rpb9 is required, along with TFIIS, to effect transcription through blocks to elongation encoded by the DNA template (12). A role in the modulation of initiation and elongation is consistent with the localization of Rpb9 in the three-dimensional structure of yeast RNA polymerase II. Rpb9 is located at the tip of the so-called "jaws" of the enzyme, which is thought to function by clamping the DNA downstream of the active site (3, 13, 14). The Rpb9 homologue in RNA polymerase III, C11, also has been implicated in regulating RNA chain elongation (15).Rpb9 comprises two zinc ribbon domains joined by a 30-amino acid l...
The role of yeast RNA polymerase II (pol II) subunit RPB9 in transcript elongation was investigated by examining the biochemical properties of pol II lacking RPB9 (pol II⌬9). The maximal rate of chain elongation was nearly identical for pol II and pol II⌬9. By contrast, pol II⌬9 elongated more efficiently through DNA sequences that signal the elongation complex to pause or arrest. The addition of purified recombinant RPB9 to pol II⌬9 restored the elongation properties of the mutant polymerase to those of the wild-type enzyme. Arrested pol II⌬9 complexes were refractory to levels of TFIIS that promoted maximal read-through with pol II. However, both pol II and pol II⌬9 complexes stimulated with TFIIS undergo transcript cleavage, confirming that transcript cleavage and read-through activities can be uncoupled. Our observations suggest that both TFIIS and RPB9 are required to stimulate the release of RNA polymerase II from the arrested state.The mRNA transcription machinery in eukaryotes is a complex of more than 30 different polypeptides of which 12 polypeptides are tightly associated with RNA polymerase (pol) 1 II. The largest two subunits of pol II, which are thought to harbor catalytic activity, are related to the largest subunits of the other nuclear RNA polymerases (1, 2). Each of the remaining pol II subunits is conserved throughout the Eukarya and more than half are also conserved in the Archae (3).In Saccharomyces cerevisiae, the genes encoding the twelve subunits of pol II have been cloned and sequenced. Five of the ten small subunits of yeast pol II (RPB5, RPB6, RPB8, RPB10, and RPB12) are common to all three nuclear RNA polymerases (4, 5). Several remaining pol II subunits have homologues in pol I and pol III. RPB11 (6) is related to AC19, a subunit shared by pols I and III (7). RPB7 is similar to the pol III subunit C25 (8). RPB9 is related to the pol I subunit, A12.2 (9). At least six pol II small subunits from S. cerevisiae are functionally interchangeable with human subunits (RPB6, RPB7, RPB8, RPB9, RPB10, and RPB12) (10 -12). Genetic analysis indicates that only two yeast pol II subunits, RPB4 and RPB9, are not essential for cell viability (13,14).In yeast, deletion of RPB9 results in mild temperature sensitivity and relatively normal levels of transcription in vivo. However, for most genes examined, the selectivity of the site of transcription initiation is altered, with new start sites shifted upstream relative to wild-type sites (15-17). The transcription initiation phenotype can be recapitulated in vitro, and addition of recombinant purified RPB9 (rRPB9) restores wild-type start site selection (15). RPB9 from S. cerevisiae is a 122-amino acid polypeptide that contains two zinc binding domains (14). The COOH-terminal zinc binding domain shares 25% sequence identity with that of the general transcript elongation factor TFIIS (18) and is predicted to adopt a zinc ribbon fold (17)(18)(19). This domain is required for the function of RPB9 in start site selection 2 and is required within TFIIS for el...
The growth of three-dimensional protein crystals is seeded by two-dimensional crystals formed on lipid layers. Such crystallization occurs faster and at lower precipitant and protein concentrations than conventional crystal growth. This approach may also allow the crystallization of proteins that resist attempts at crystal growth by other means.
The RPB9 subunit of RNA polymerase II regulates transcription elongation activity and is required for the action of the transcription elongation factor, TFIIS. RPB9 comprises two zinc ribbon domains joined by a conserved linker region. The C-terminal zinc ribbon is similar in sequence to that found in TFIIS. To elucidate the relationship between the structure and transcription elongation function of RPB9, we initiated a mutagenesis study on the Saccharomyces cerevisiae homologue. The individual zinc ribbon domains, in isolation or in combination, could not stimulate transcription by a polymerase lacking RPB9, pol II⌬9. Mutations in the N-terminal zinc ribbon had little effect on transcription activity. By contrast, mutations in the acidic loop that connects the second and third -strands of the C-terminal zinc ribbon were completely inactive for transcription. Interestingly, the analogous residues in TFIIS are also critical for elongation activity. A conserved charged stretch in the linker region (residues 89 -95, DPTLPR) mediated the interaction with RNA polymerase II.The eukaryotic transcription elongation factor TFIIS stimulates RNA polymerase II to cleave the 3Ј end of nascent transcripts, which in turn promotes transcription through blocks to elongation. TFIIS homologues have been identified in species ranging from yeast to man. The protein is composed of three structurally independent domains (I, II, and III) (1), which comprise a four-helix bundle, a three-helix bundle, and a zinc ribbon domain, respectively (1-4). The central three-helix bundle mediates binding to RNA polymerase II (5), whereas residues within the zinc ribbon region are important for transcript cleavage (6).The zinc ribbon is defined by a conserved zinc-chelating motif, CX 2 CX 24 CX 2 C, that forms a three-stranded anti-parallel -sheet with the four cysteines coordinating the zinc ion (2, 4). Although first identified in TFIIS, sequence and structural analysis studies have suggested that zinc ribbons are present in many eukaryotic transcription-associated proteins, including transcription factors TFIIB and TFIIE, as well as RNA polymerase II subunits RPB1, RPB2, RPB12, and RPB9 (7,8). RPB9 is composed primarily of two zinc-binding regions, which are defined by the CX 2 CX n CX 2 C motif. The C-terminal zinc domain is very similar (30% identity) to the zinc ribbon domain from TFIIS (9). RPB9 is highly conserved between yeast (10), archaebacteria (9, 11), Drosophila (12), plants (13), and humans (14). In addition, analogous subunits have been identified as components of the eukaryotic RNA polymerases I and III (15).1 The high degree of conservation of this subunit is underlined by the ability of the human homologue to partially substitute for yeast subunit in vivo (17). However, the functional conservation is not universal; Drosophila RPB9 is not functional in the yeast transcription system (13).The conservation of the zinc ribbon structural motif between TFIIS and RPB9 extends to function. RPB9 is required for TFIIS to stimulate elongat...
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