Role of a small RNA pol II subunit in TATA to transcription start site spacing

To assess functional relatedness of individual components of the eukaryotic transcription apparatus, three human subunits (hsRPB5, hsRPB8, and hsRPB10) were tested for their ability to support yeast cell growth in the absence of their essential yeast homologs. Two of the three subunits, hsRPB8 and hsRPB10, supported normal yeast cell growth at moderate temperatures. A fourth human subunit, hsRPB9, is a homolog of the nonessential yeast subunit RPB9. Yeast cells lacking RPB9 are unable to grow at high and low temperatures and are defective in mRNA start site selection. We tested the ability of hsRPB9 to correct the growth and start site selection defect seen in the absence of RPB9. Expression of hsRPB9 on a high-copy-number plasmid, but not a low-copy-number plasmid, restored growth at high temperatures. Recombinant human hsRPB9 was also able to completely correct the start site selection defect seen at the CYC1 promoter in vitro as effectively as the yeast RPB9 subunit. Immunoprecipitation of the cell extracts from yeast cells containing either of the human subunits that function in place of their yeast counterparts in vivo suggested that they assemble with the complete set of yeast RNA polymerase II subunits. Overall, a total of six of the seven human subunits tested previously or in this study are able to substitute for their yeast counterparts in vivo, underscoring the remarkable similarities between the transcriptional machineries of lower and higher eukaryotes.The eukaryotic mRNA transcription apparatus comprises RNA polymerase II, general transcription factors and their associated factors, and gene-specific factors (reviewed in references 6, 13, and 23). RNA polymerase II (pol II) is a multisubunit enzyme with ϳ12 to 15 subunits, depending on the organism, that plays a major role in mRNA synthesis since it possesses the catalytic machinery for the formation of the 3Ј-5Ј phosphodiester bonds between ribonucleoside triphosphates and presumably responds to signals from the multiple factors involved in regulating its function during initiation and elongation of mRNA synthesis.Yeast Saccharomyces cerevisiae pol II has been a useful paradigm for enzyme function since its subunit structure and amino acid sequences are strikingly similar to pol II subunits from a variety of eukaryotes and even archaebacteria. S. cerevisiae pol II has 12 subunits, designated RPB1-RPB12, ranging in size from ϳ190 to ϳ8 kDa (reviewed in references 26 and 27). Five of these subunits (RPB5, RPB6, RPB8, RPB10, and RPB12) are also present in RNA polymerases I and III and are usually referred to as the common subunits.Since the functions of most pol II subunits are unclear, one approach to understanding their role in transcription has been to isolate the genes encoding human RNA polymerase subunits and test for functional similarities by determining if the human subunit can function in place of its yeast homolog. Only a few of the human subunit genes have been tested for heterocomplementation in S. cerevisiae. hsRPB6 has a highly conserve...

Section: Resultssupporting

confidence: 87%

Six Human RNA Polymerase Subunits Functionally Substitute for Their Yeast Counterparts

McKune

Moore

Hull

et al. 1995

“…To further examine the start site shift, we studied initiation in a strain of yeast deleted for RPB9. This small subunit of RNA polymerase II is not essential, but its loss shifts transcription start sites upstream and impairs elongation more generally (2,14,18,19,45). It also renders cells extremely MPA sensitive as well as defective in IMD2 induction (39).…”

Section: Resultsmentioning

confidence: 99%

“…RNA polymerase II without this subunit initiates at the upstream site but cannot initiate from the A start downstream. This makes sense, since Rpb9 is known to play a role in start site positioning generally and is a significant component of one of the pincers of the enzyme's jaws that contacts downstream duplex DNA (9,14,18,19). Absence of the subunit may result in a structural deficit, such as a loose grip on DNA, that hampers controlled translocation of the enzyme.…”

Section: Discussionmentioning

confidence: 99%

Properties of an Intergenic Terminator and Start Site Switch That Regulate IMD2 Transcription in Yeast

Jenks

O'Rourke

Reines

2008

The IMD2 gene in Saccharomyces cerevisiae is regulated by intracellular guanine nucleotides. Regulation is exerted through the choice of alternative transcription start sites that results in synthesis of either an unstable short transcript terminating upstream of the start codon or a full-length productive IMD2 mRNA. Start site selection is dictated by the intracellular guanine nucleotide levels. Here we have mapped the polyadenylation sites of the upstream, unstable short transcripts that form a heterogeneous family of RNAs of Ϸ200 nucleotides. The switch from the upstream to downstream start sites required the Rpb9 subunit of RNA polymerase II. The enzyme's ability to locate the downstream initiation site decreased exponentially as the start was moved downstream from the TATA box. This suggests that RNA polymerase II's pincer grip is important as it slides on DNA in search of a start site. Exosome degradation of the upstream transcripts was highly dependent upon the distance between the terminator and promoter. Similarly, termination was dependent upon the Sen1 helicase when close to the promoter. These findings extend the emerging concept that distinct modes of termination by RNA polymerase II exist and that the distance of the terminator from the promoter, as well as its sequence, is important for the pathway chosen.

“…Genetic analysis has identified mutations in TFIIB, and three subunits of RNAPII, RPB1, RPB2, and RPB9, which alter transcription start site selection in yeast cells in vivo (4,17,21,23,44). Interestingly, mutations in the large subunit of TFIIF are able to compensate for mutations in TFIIB and restore the correct position of transcription initiation (58).…”

Section: Discussionmentioning

confidence: 99%

Accurate Positioning of RNA Polymerase II on a Natural TATA-Less Promoter Is Independent of TATA-Binding-Protein-Associated Factors and Initiator-Binding Proteins

Weis

Reinberg

1997

Two promoter elements, the TATA element and initiator (Inr), are capable of directing specific transcription initiation of protein-encoding genes by RNA polymerase II (RNAPII). Although binding to the TATA element by the TATA-binding protein (TBP) has been shown to be the initial recognition step in transcription complex formation in vitro, the mechanism through which the basal machinery assembles into a functional complex on TATA-less promoters is controversial. Evidence supporting numerous models of Inr-mediated transcription complex formation exists, including the nucleation of a complex by Inr-binding proteins, a component of the TFIID complex, or a specific upstream activator common to many TATA-less promoters, Sp1. Using various techniques, we have undertaken a systematic analysis of the natural TATA-less human DNA polymerase ␤ (␤-pol) gene promoter. Although the ␤-pol promoter contains upstream Sp1 elements and a functional Inr that binds YY1, neither of these factors is essential for Inr-mediated transcription complex formation. A complex containing TBP, TFIIB, TFIIF, and RNAPII (DBPolF complex) is capable of forming on the promoter in an Inr-dependent manner. A single point mutation within the Inr that affects DBPolF complex formation diminishes ␤-pol transcriptional activity.Transcription initiation of protein-encoding genes is a complex process requiring the precise positioning of RNA polymerase II (RNAPII) on promoter DNA. This is accomplished via a series of interactions between RNAPII and at least six accessory proteins, TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH, termed the general transcription factors (GTFs) (37, 70). Additionally, nucleation of an initiation complex occurs by recognition of a specific core element (31,54,66). In many genes, the TATA element is the primary core element responsible for positioning the basal transcription machinery on the promoter. TFIID, a multisubunit protein complex consisting of the TATA-binding protein (TBP) and TBP-associated factors (TAFs), nucleates the formation of the transcription initiation complex by binding to the TATA element (8,22,59). In vitro experiments have shown that preinitiation complex assembly proceeds by sequential recruitment of TFIIB followed by RNAPII together with TFIIF, TFIIE, and TFIIH to the promoter-bound TFIID. This multistep model has recently been challenged by the isolation of an RNAPII complex containing a subset of the GTFs, SRB (suppressor of RNAP B) proteins, and additional polypeptides capable of interacting with each other in the absence of promoter DNA (29). Under the appropriate conditions, this complex is capable of specifically initiating transcription and mediating a response to transcriptional activators in vitro (30, 38).As more promoters of protein-encoding genes have been characterized, it has been found that many lack a TATA element (31,54,66). Studies using the TATA-less murine terminal deoxynucleotidyltransferase (TdT) promoter identified a second core element, the initiator (Inr), which encompasses the tra...