RNA polymerase III transcription complexes on chromosomal 5S rRNA genes in vivo: TFIIIB occupancy and promoter opening.

Quantitative analysis of multiple-hit potassium permanganate (KMnO(4)) footprinting has been carried out in vivo on Saccharomyces cerevisiae 5S rRNA genes. The results fix the number of open complexes at steady state in exponentially growing cells at between 8 and 17% of the 150 to 200 chromosomal copies. UV and dimethyl sulfate footprinting set the transcription factor TFIIIB occupancy at 23 to 47%. The comparison between the two values suggests that RNA polymerase III binding or promoter opening is the rate-limiting step in 5S rRNA transcription in vivo. Inhibition of RNA elongation in vivo by cordycepin confirms this result. An experimental system that is capable of providing information on the mechanistic steps involved in regulatory events in S. cerevisiae cells has been established.

Assembly of 5S ribosomal RNA is required at a specific step of the pre-rRNA processing pathway.

A collection of yeast strains surviving with mutant 5S RNA has been constructed. The mutant strains presented alterations of the nucleolar structure, with less granular component, and a delocalization of the 25S rRNA throughout the nucleoplasm. The 5S RNA mutations affected helix I and resulted in decreased amounts of stable 5S RNA and of the ribosomal 60S subunits. The shortage of 60S subunits was due to a specific defect in the processing of the 27SB precursor RNA that gives rise to the mature 25S and 5.8S rRNA. The processing rate of the 27SB pre-rRNA was specifically delayed, whereas the 27SA and 20S pre-rRNA were processed at a normal rate. The defect was partially corrected by increasing the amount of mutant 5S RNA. We propose that the 5S RNA is recruited by the pre-60S particle and that its recruitment is necessary for the efficient processing of the 27SB RNA precursor. Such a mechanism could ensure that all newly formed mature 60S subunits contain stoichiometric amounts of the three rRNA components.

The only essential function of TFIIIA in yeast is the transcription of 5S rRNA genes.

We have developed a system to transcribe the yeast 5S rRNA gene in the absence of the transcription factor TFIIIA. A long transcript was synthesized both in vitro and in vivo from a hybrid gene in which the tRNA-like promoter sequence of the RPR1 gene was fused to the yeast 5S RNA gene. No internal initiation directed by the endogenous 5S rDNA promoter or any processing of the hybrid transcript was observed in vitro. Yeast cells devoid of transcription factor TFIIIA, which, therefore, could not synthesize any 5S rRNA from the endogenous chromosomal copies of 5S rDNA, could survive if they carried the hybrid RPR1-5S construct on a multicopy plasmid. In this case, the only source of 5S rRNA was the precursor RPR1-5S transcript that gave rise to two RNA species slightly larger than wild-type 5S rRNA. This establishes that the only essential function of TFIIIA is to promote the synthesis of 5S rRNA. However, cells devoid of TFIIIA and surviving with these two RNAs grew more slowly at 30 degrees C compared with wild-type cells and were thermosensitive at 37 degrees C.

Identification of potential target genes for Adr1p through characterization of essential nucleotides in UAS1.

Adr1p is a regulatory protein in the yeast Saccharomyces cerevisiae that binds to and activates transcription from two sites in a perfect 22-bp inverted repeat, UAS1, in the ADH2 promoter. Binding requires two C2H2 zinc fingers and a region amino terminal to the fingers. The importance for DNA binding of each position within UAS1 was deduced from two types of assays. Both methods led to an identical consensus sequence containing only four essential base pairs: GG(A/G)G. The preferred sequence, TTGG(A/G)GA, is found in both halves of the inverted repeat. The region of Adr1p amino terminal to the fingers is important for phosphate contacts in the central region of UAS1. However, no base-specific contacts in this portion of UAS1 are important for DNA binding or for ADR1-dependent transcription in vivo. When the central 6 bp were deleted, only a single monomer of Adr1p was able to bind in vitro and activation in vivo was severely reduced. On the basis of these results and previous knowledge about the DNA binding site requirements, including constraints on the spacing and orientation of sites that affect activation in vivo, a consensus binding site for Adr1p was derived. By using this consensus site, potential Adr1p binding sites were located in the promoters of genes known to show ADR1-dependent expression. In addition, this consensus allowed the identification of new potential target genes for Adr1p.

TATA box-binding protein (TBP) is a constituent of the polymerase I-specific transcription initiation factor TIF-IB (SL1) bound to the rRNA promoter and shows differential sensitivity to TBP-directed reagents in polymerase I, II, and III transcription factors.

The role of the Acanthamoeba castellanii TATA-binding protein (TBP) in transcription was examined. Specific antibodies against the nonconserved N-terminal domain of TBP were used to verify the presence of TBP in the fundamental transcription initiation factor for RNA polymerase I, TIF-IB, and to demonstrate that TBP is part of the committed initiation complex on the rRNA promoter. The same antibodies inhibit transcription in all three polymerase systems, but they do so differentially. Oligonucleotide competitors were used to evaluate the accessibility of the TATA-binding site in TIF-IB, TFIID, and TFIIIB. The results suggest that insertion of TBP into the polymerase II and III factors is more similar than insertion into the polymerase I factor.

ADH2 expression is repressed by REG1 independently of mutations that alter the phosphorylation of the yeast transcription factor ADR1.

In Saccharomyces cerevisiae, expression of the ADH2 gene is undetectable during growth on glucose. The transcription factor ADR1 is required to fully activate expression when glucose becomes depleted. Partial activation during growth on glucose occurred in cells carrying a constitutive allele of ADR1 in which the phosphorylatable serine of a cyclic AMP (cAMP)-dependent protein kinase phosphorylation site had been changed to alanine. When glucose was removed from the growth medium, a substantial increase in the level of this constitutive expression was observed for both the ADH2 gene and a reporter construct containing the ADR1 binding site. This suggests that glucose can block ADR1-mediated activation independently of cAMP-dependent phosphorylation at serine 230. REG1/HEX2/SRN1 was identified as a potential serine 230-independent repressor of ADH2 expression. Yeast strains carrying a deletion of the REG1 gene, reg1-1966, showed a large increase in ADR1-dependent expression of ADH2 during growth on glucose. A smaller increase in ADR1-independent expression was also observed. Additionally, an increase in the level of ADR1 expression and posttranslational modification of the ADR1 protein were observed. When the reg1-1966 allele was combined with various ADR1 constitutive alleles, the level of ADH2 expression was synergistically elevated. This indicates that REG1 can act independently of phosphorylation at serine 230. Our results suggest that glucose repression in the presence of ADR1 constitutive alleles occurs primarily through a REG1-dependent pathway which appears to affect ADH2 transcription at multiple levels.

A mutation outside the two zinc fingers of ADR1 can suppress defects in either finger.

A second-site mutation that restored DNA binding to ADR1 mutants altered at different positions in the two zinc fingers was identified. This mutation (called IS1) was a conservative change of arginine 91 to lysine in a region amino terminal to the two zinc fingers and known from previous experiments to be necessary for DNA binding. IS1 increased binding to the UAS1 sequence two- to sevenfold for various ADR1 mutants and twofold for wild-type ADR1. The change of arginine 91 to glycine decreased binding twofold, suggesting that this arginine is involved in DNA binding in the wild-type protein. The increase in binding by IS1 did not involve protein-protein interactions between the two ADR1 monomers, nor did it require the presence of the sequences flanking UAS1. However, the effect of IS1 was influenced by the sequence of the first finger, suggesting that interactions between the region amino terminal to the fingers and the fingers themselves could exist. A model for the role of the amino-terminal region based on these results and sequence homologies with other DNA-binding motifs is proposed.

Inactivation of the protein phosphatase 2A regulatory subunit A results in morphological and transcriptional defects in Saccharomyces cerevisiae.

We have determined that TPD3, a gene previously identified in a screen for mutants defective in tRNA biosynthesis, most likely encodes the A regulatory subunit of the major protein phosphatase 2A species in the yeast Saccharomyces cerevisiae. The predicted amino acid sequence of the product of TPD3 is highly homologous to the sequence of the mammalian A subunit of protein phosphatase 2A. In addition, antibodies raised against Tpd3p specifically precipitate a significant fraction of the protein phosphatase 2A activity in the cell, and extracts of tpd3 strains yield a different chromatographic profile of protein phosphatase 2A than do extracts of isogenic TPD3 strains. tpd3 deletion strains generally grow poorly and have at least two distinct phenotypes. At reduced temperatures, tpd3 strains appear to be defective in cytokinesis, since most cells become multibudded and multinucleate following a shift to 13 degrees C. This is similar to the phenotype obtained by overexpression of the protein phosphatase 2A catalytic subunit or by loss of CDC55, a gene that encodes a protein with homology to a second regulatory subunit of protein phosphatase 2A. At elevated temperatures, tpd3 strains are defective in transcription by RNA polymerase III. Consistent with this in vivo phenotype, extracts of tpd3 strains fail to support in vitro transcription of tRNA genes, a defect that can be reversed by addition of either purified RNA polymerase III or TFIIIB. These results reinforce the notion that protein phosphatase 2A affects a variety of biological processes in the cell and provide an initial identification of critical substrates for this phosphatase.

On the flexible interaction of yeast factor tau with the bipartite promoter of tRNA genes.

Yeast transcription factor tau (analogous to vertebrate TFIIIC) interacts specifically with the internal split promoter of tRNA genes. Binding to the two promoter elements (A block and B block) occurs within 30 seconds even when they are separated by a long intervening sequence. Dimethylsulfate protection analysis of contact points between tau and the noncoding strand of a series of internally deleted tRNA3(Leu) genes shows that the specificity of the interaction is not affected by changes in the distance or in the relative helical orientation of the promoter elements. This result is consistent with the results of previous footprinting experiments (Baker, R.E., Camier, S., Sentenac, A. and Hall, B.D., 1987, Proc. Natl. Acad. Sci. USA, 84, 8768-8772). To test if any physical constraint is imposed on the DNA molecule upon tau binding, we analyzed the effect of introducing random single-strand breaks in the noncoding strand of the tRNA gene. Whereas some nicks located in the A block were found to prevent tau binding, no single-strand break in the B block region or in the DNA between the A and B blocks were observed to inhibit or facilitate the binding of tau. We therefore propose that the great flexibility of the tau-tDNA interaction is mostly due to the tau protein itself.

The U6 gene of Saccharomyces cerevisiae is transcribed by RNA polymerase C (III) in vivo and in vitro.

Unlike the majority of genes encoding small nuclear RNAs, which are transcribed by RNA polymerase B, the U6 gene contains features found in both class B and class C genes, indicating the involvement of a combination of transcription factors normally specific to each class of genes. We present direct genetic and biochemical evidence that the U6 gene of Saccharomyces cerevisiae is transcribed by RNA polymerase C in vivo as well as in vitro. A mutant strain with a temperature-sensitive defect in the large subunit of RNA polymerase C that results in defective transcription of tRNA and 5S RNA genes shows a corresponding defect in U6 RNA levels. Also, purified RNA polymerase C transcribes the U6 gene when supplemented with partially purified TFIIIB. The other class C transcription factors, TFIIIA and Tau (TFIIIC), are not required in this system.

Gene size differentially affects the binding of yeast transcription factor tau to two intragenic regions.

Yeast transcription factor tau (transcription factor IIIC) specifically interacts with tRNA genes, binding to both the A block and the B block elements of the internal promoter. To study the influence of A block-B block spacing, we analyzed the binding of purified tau protein to a series of internally deleted yeast tRNA(3Leu) genes with A and B blocks separated by 0 to 74 base pairs. Optimal binding occurred with genes having A block-B block distances of 30-60 base pairs; the relative helical orientation of the A and B blocks was unimportant. Results from DNase I "footprinting" and lambda exonuclease protection experiments were consistent with these findings and further revealed that changes in A block-B block distance primarily affect the ability of tau to interact with A block sequences; B block interactions are unaltered. When the A block-B block distance is 17 base pairs or less, tau interacts with a sequence located 15 base pairs upstream of the normal A block, and a new RNA initiation site is observed by in vitro transcription. We propose that the initial binding of tau to the B block activates transcription by enhancing its ability to bind at the A block, and that the A block interaction ultimately directs initiation by RNA polymerase III.

Selective proteolysis defines two DNA binding domains in yeast transcription factor tau.

Transcription of eukaryotic transfer RNA genes involves, as a primary event, the stable binding of a protein factor to the intragenic promoter. The internal control region is composed of two non-contiguous conserved sequence elements, the A and B blocks. These are variably spaced depending on the genes. tau, a large transcription factor purified from yeast cells, interacts with these two control elements as shown by DNase I footprinting, exonuclease digestion, dimethyl sulphate protection experiments and by analysis of point mutations. Here we used a limited proteolysis treatment to obtain a smaller form of tau with drastically altered DNA binding properties. A protease-resistant domain interacts solely with the B block region of tRNA genes.

A split binding site for transcription factor tau on the tRNA3Glu gene.

Yeast transcription factor tau forms a stable complex with tRNA genes. Using this property, the factor could be highly purified on a specific tDNA column. The purified factor was found by DNA footprinting to protect the whole yeast tRNA3Glu gene from position -8 to +81. A DNase-sensitive site was retained in the middle of the gene on both strands. The 3' border of the complex was mapped by exonuclease digestion at +88, just downstream of the termination signal. The 5' limit of the complex was found at position -11. However, upon prolonged incubation with exonuclease, the -11 blockage disappeared and the DNA molecules were digested to position +30 to 38 in the middle of the gene. Contact points at guanine residues were identified by dimethyl sulphate protection experiments. Reduced methylation of G residues in the presence of factor was found solely within the A block and in the B block region. All six invariant GC pairs (i.e., G10, G18, G19 and G53, C56 and C61) were found to have strong contacts with the factor. These results show that tau factor interacts with both the 5' and 3' half of the tRNA3Glu gene, with the B block region being the predominant binding site. The presence of this dual binding site suggests a model in which the factor would bind alternately at the A and B block regions to allow transcription of the internal promoter by RNA polymerase C.

Isolation of a class C transcription factor which forms a stable complex with tRNA genes.

A yeast extract was fractionated to resolve the factors involved in the transcription of yeast tRNA genes. An in vitro transcription system was reconstituted with two separate protein fractions and purified RNA polymerase C (III). Optimal conditions for tRNA synthesis have been determined. One essential component, termed tau factor, was partially purified by conventional chromatographic methods on heparin-agarose and DEAE-Sephadex; it sedimented as a large macromolecule in glycerol gradients (mol. wt. approximately 300 000). tau factor was found to form a stable complex with the tRNA gene in the absence of other transcriptional components. Complex formation is very fast, is not temperature dependent between 10 degrees C and 25 degrees C and does not require divalent cations. The factor-DNA complex is stable for at least 30 min at high salt concentration (0.1 M ammonium sulfate). These results indicate that gene recognition by a specific factor is a primary event in tRNA synthesis.

Stimulation of transcription of the yeast tRNATyr gene in cell-free extracts by tyrosyl-tRNA synthetase.

Eukaryotic transfer RNA genes have two internal discontinuous control regions, the A and B blocks, which correspond approximately to the D-stem and the pseudo-U arm of tRNA. In reconstituted transcription systems at least two components are required to direct accurate initiation by RNA polymerase (refs 4,5). However, little is known about the mechanism of interaction of the internal promoter sequences with factors and RNA polymerase C within the transcription complex, although tRNA-like conformation of the B block sequence was surmised to be critical for DNA recognition. By analogy with the 5S RNA system, where a transcription factor required for 5S DNA expression was shown to interact both with 5S RNA and with the noncoding strand of the 5S gene, we explored the possibility that a protein which normally binds to tRNA could also interact with the tRNA gene and regulate its transcription. Here we show that in vitro transcription of the yeast SUP4 tRNATyr gene in crude yeast extracts is strongly stimulated by tyrosyl-tRNA synthetase (TyrRS) but not by two other non-cognate synthetases. Substrates of the synthetase, tRNATyr and tyrosine, interfere with stimulation of tRNA synthesis.