TATA binding protein can stimulate core-directed transcription by yeast RNA polymerase I

Abstract

The TATA binding protein (TBP) interacts with two transcription factor complexes, upstream activating factor (UAF) and core factor (CF), to direct transcription by RNA polymerase I (polI) in the yeast Saccharomyces cerevisiae. Previous work indicates that one function of TBP is to serve as a bridge, enabling UAF to recruit and stabilize the binding of CF (23, 24). In this work we show that, in addition to aiding recruitment, TBP also directly aids CF function. Overexpression of TBP in strains with UAF components deleted will stimulate CF-directed transcription nearly to wild-type levels in vivo. In vitro, increasing the concentration of TBP stimulates CF-directed transcription in the absence of either UAF or its DNA binding site. This dual function of TBP, serving as a critical member of a core promoter complex as well as a contact point for upstream activators, appears similar to the dual roles that TBP also plays in transcription by RNA polII.

FIG. 1

Overexpression of TBP bypasses the requirement for UAF components in vivo. (A) Strains carrying disruptions of any of the CF subunits (ΔRRN6, ΔRRN7, and ΔRRN11) are able to grow on galactose (left plate) due to rRNA transcribed from the GAL7 promoter on pNOY103 but are unable to grow on glucose (right plate). Overexpression of TBP (top sectors) does not alter this phenotype (compare to bottom sectors). (B) Strains carrying disruptions of UAF subunits (ΔRRN5, ΔRRN9, and ΔRRN10) also grow on galactose due to rRNA transcribed from pNOY103 (left plate). On glucose the UAF disruption strains grow very slowly (right plate, bottom sectors). However, overexpression of TBP allows the UAF disruption strains to grow at near wild-type rates (right plate, top sectors). This result indicates that TBP overexpression bypasses the requirement for the UAF complex.

FIG. 2

Transformation with a TBP expression vector (pSH223) results in overexpression of the protein. Protein extracts from various strains were blotted onto nitrocellulose and probed with a polyclonal antibody against yeast TBP. As a loading control the same blots were also probed with an antibody against the polII transcription factor TFIIH. Lane 1, extract from wild-type (wt) cells (strain RLY01); lane 2, wild type transformed with pSH223; lane 3, strain disrupted in RRN5 (RLY07); lane 4, ΔRRN5 strain transformed with pSH223. Transformation with pSH223 increases TBP levels significantly in either wild-type or ΔRRN5 cells (lanes 3 and 4) without affecting the level of TFIIH.

FIG. 3

Overexpression of TBP stimulates rRNA transcription in a strain with an inactive UAF (ΔRRN5). Newly made rRNAs in various strains were pulse-labeled and separated by gel electrophoresis, and the radioactive bands were detected by fluorography. The positions of mature 18S and 25S rRNAs are indicated. TBP overexpression has no effect on rRNA synthesis in a wild-type (wt) strain (RLY01, compare lanes 1 and 2). In contrast, TBP overexpression in a ΔRRN5 strain stimulates rRNA synthesis (RLY07) (lanes 3 and 4). As expected, no rRNA synthesis is detected in a ΔRRN11 strain, with or without TBP overexpression (RLY02) (lanes 5 and 6).

FIG. 4

TBP overexpression stimulates rRNA production by polI. Strain RLY07 (ΔRRN5) was secondarily transformed with either an empty vector (pRS315), a vector expressing RRN5 (pRS315RRN5), or the TBP overexpression vector (pSH223). In parallel, strain RLY4811 (isogenic to RLY07 but also carrying a temperature-sensitive [ts] allele of RPA190) was transformed with the same three vectors. (A) At the permissive temperature (30°C) strains RLY07 and RLY4811 behave the same. They grow on galactose due to the presence of pNOY103 and fail to grow on glucose. Transformation with an expression vector for either RRN5 or TBP allows both strains to grow on glucose. (B) At the nonpermissive temperature (35.5°C) strain RLY4811 cannot grow on glucose even when RRN5 or TBP is expressed (right plate, bottom sectors). This result indicates that TBP overexpression stimulates rRNA production via polI and not via some other RNA polymerase.

FIG. 5

Requirement for either the upstream domain of the polI promoter or the UAF complex can be bypassed in vitro by overexpression of TBP. (A) Transcription extracts were made from either wild-type (WT) (RLY01) cells (lanes 1 through 6) or from wild-type cells overexpressing TBP (lanes 7 through 12). Each extract was used to transcribe templates bearing either a wild-type polI promoter (lanes 1, 4, 7, and 10), a promoter with an inactivated upstream domain (LS_up; lanes 2, 5, 8, and 11), or a promoter with an inactivated core domain (LS_core; lanes 3, 6, 9, and 12). Assays were performed at either a high (10-μg/ml) or a low (0.5-μg/ml) template concentration. Each extract was titrated to determine the amount which gave the maximum transcription with a wild-type template at 10 μg of DNA per ml. Thirty percent of the maximal amount of extract was used for each of the experiments whose results are shown. In wild-type extracts, the negative effect of inactivating the upstream domain (LS_up) can be overcome by increasing the template concentration (compare lanes 2 and 5). Inactivation of the core domain (LS_core) eliminates transcription at all template concentrations (lanes 3 and 6). Overexpression of TBP strongly stimulates transcription from a template lacking an upstream domain at a low template concentration (compare lanes 5 and 11). (B) Extracts were made from a ΔRRN5 strain (RLY07) or from a ΔRRN5 strain overexpressing TBP. Each extract was used to transcribe either a wild-type template (lanes 1 and 5), a template with a 5′ deletion to position −122 of the promoter (lanes 2 and 6), a template deleted to −42 (lanes 3 and 7), or a template deleted to −2 (lanes 4 and 8) at a 10-μg/ml template concentration. Extracts were used at 30% of the amount which gave maximal activity on a wild-type template. Overexpressing TBP stimulates transcription from promoters bearing deletions of the UAF binding region (lanes 6 and 7) to the same level seen with an intact promoter (lane 5).

FIG. 6

Recombinant TBP stimulates transcription from the core domain in an extract defective for UAF. Extract from strain RLY07 was used to transcribe templates either mutated in the upstream domain (LS_up, lanes 1 through 4), mutated in the core domain (LS_core, lanes 5 and 6), or having the upstream domain deleted (Δ−42, lanes 7 through 11). Template concentrations were 10 μg/ml, and recombinant TBP (rTBP) was added to the reaction mixtures as indicated. PhosphorImager quantitation indicates about a threefold stimulation of transcription between lanes 1 and 3 and about a fourfold stimulation between lanes 7 and 10.