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A new in vitro approach is used to investigate the specificity of purified yeast RNA polymerase B (II). The template is supercoiled, the transcription is primed by a dinucleotide, and the transcripts are analyzed by polyacrylamide gel electrophoresis after synthesis in the absence of one nucleoside triphosphate. Under these conditions, two recombinant plasmids carrying the gene or part of the gene for yeast alcohol dehydrogenase I direct the synthesis of a very limited number of oligonucleotides. Elongation of these prelabeled oligomers, using unlabeled substrates, occurs in a discontinuous way. A major transcript of 200 nucleotides accumulates transiently. Southern hybridization shows that it is initiated about 1,400 bases upstream from the origin of the yeast alcohol dehydrogenase I gene. A minor start was identified, by a modified runoff experiment, at position -35 from the AUG initiation codon. The location of this site is related to the presumptive in vivo transcription starts. The selectivity disappears when the template is a truncated DNA. Then, initiation occurs predominantly at nicks introduced by the restriction enzymes.

One gene coding for yeast cytoplasmic elongation factor 1 alpha (EF-1 alpha) was isolated by colony hybridization using a cDNA probe prepared from purified EF-1 alpha mRNA. A recombinant plasmid, pLB1, with a 6-kilobase yeast DNA insert, was found by hybrid selection and translation experiments to carry the entire gene. The nucleotide sequence of the gene with its 5'- and 3'-flanking regions was determined. The 5' and 3' ends of EF-1 alpha mRNA were localized by the S1 nuclease mapping technique. The cloned gene, called TEF1, encodes a protein of 458 amino acids (Mr = 50,071) in a single, uninterrupted reading frame. The amino acid sequence shows a strong homology with several domains of Artemia salina EF-1 alpha cytoplasmic factor, as evidenced by diagonal dot matrix analysis. Protein sequence homology is comparatively much lower with the yeast mitochondrial elongation factor. S1 nuclease mapping of the mRNA, hybridization analysis of chromosomal DNA using intragenic or extragenic DNA probes, and gene disruption experiments demonstrated the existence of two genes coding for the cytoplasmic elongation factor EF-1 alpha/haploid genome. The presence of an intact chromosomal TEF1 gene is not essential for growth of haploid yeast cells.

Fractionation of yeast extracts on heparin-agarose revealed the presence of a DNA footprinting activity that interacted specifically with the 5'-upstream region of TEF1 and TEF2 genes coding for the protein synthesis elongation factor EF-1 alpha, and of the ribosomal protein gene RP51A. The protected regions encompassed the conserved sequences 'HOMOL1' (AACATC TA CG T A G CA) or RPG-box (ACCCATACATT TA) previously detected 200-400 bp upstream of most of the yeast ribosomal protein genes examined. Two types of protein-DNA complexes were separated by a gel electrophoresis retardation assay. Complex 1, formed on TEF1, TEF2 and RP51A 5'-flanking region, was correlated with the protection of a 25-bp sequence. Complex 2, formed on TEF2 or RP51A probes at higher protein concentrations, corresponded to an extended footprint of 35-40 bp. The migration characteristics of the protein-DNA complexes and competition experiments indicated that the same component(s) interacted with the three different promoters. It is suggested that this DNA factor(s) is required for activation and coordinated regulation of the whole family of genes coding for the translational apparatus.

Yeast RNA polymerase C purified by a simple large scale method was resolved into multiple components by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Specific antibodies directed against each polypeptide chain were prepared in rabbits and used as structural and functional probes. With minor exceptions, each antibody recognized specifically the corresponding polypeptide by blot-immunodetection. Cross-reactions with purified RNA polymerases A and B confirmed our previous description of the subunits shared by the three nuclear RNA polymerases. Immunoadsorption of RNA polymerase C at different stages of purification using antibodies to subunits C160 and C128 yielded the same collection of polypeptides as found in the purified enzyme: C160, C128, C82, C53, C40, C37, C34, C31, C27, C25, C23, C19, C14.5, C12.5, and C10. Subunit-specific antibodies were used to probe the activity of RNA polymerase C in a specific, reconstituted transcription system as well as on a nonspecific template. Transcription of the tRNAGlu3 gene in vitro was inhibited when RNA polymerase C was preincubated with antibodies directed to C128, C82, C53, C34, C23, or C19. Antibodies to C82, C53, and C34 were much less inhibitory in the nonspecific assay. Inhibition by anti-C128 or anti-C23 was relieved by preincubation of enzyme C with plasmid DNA prior to antibody addition. These results are discussed in terms of the participation of these polypeptides to the active enzyme molecule, and of their possible role in DNA binding or transcription factor recognition.

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.

A lambda gt11 yeast genomic library was screened with antibodies directed against yeast RNA polymerases A, B, and C. Thirty-five individual recombinant phages that expressed proteins in Escherichia coli that were antigenically related to RNA polymerases A, B, or C were isolated by using 22 distinct antisera. Thus, all 22 genes for the RNA polymerase subunits were potentially cloned. In three cases (lambda A-43, lambda A-40, and lambda A-34.5), an antigenic protein was expressed in E. coli with the same molecular weight as the corresponding subunit. When lambda A-40 DNA was used to hybrid-select yeast mRNA, the protein translated in vitro was the expected size for the A-40 subunit, further supporting our isolation of the A-40 gene. However, mRNA hybrid selected by lambda A-27 DNA did not code for a protein of the correct size. The lengths of the mRNA that hybridized to phage lambda A-190 or lambda C-160 DNA on RNA blots were in agreement with the predicted sizes of the coding regions of the corresponding genes. As predicted by our previous immunological results, yeast DNA inserts of the lambda A-190 and lambda C-160 clones cross-hybridized to the B-220 subunit gene. The cloned genes for the RNA polymerase subunits will prove to be valuable tools for the study of the function, regulation, and genetics of the yeast RNA polymerases.

Yeast RNA polymerase B catalyzes an efficient abortive initiation on double-stranded DNA templates using the appropriate combination of primer and substrate. The specificity of initiation was investigated using a recombinant plasmid (pJD14 DNA) containing the structural gene for yeast alcohol dehydrogenase I (ADHI). The combination of the dinucleotide UpA and UTP was 10 fold more efficient with pJD14 DNA than with the vector pBR322 DNA to direct the synthesis of the trinucleotide UpApU. Under these conditions, stable enzyme-DNA complexes were formed and could be retained on nitrocellulose filters. Using the UpA-primed system and a short pulse of RNA synthesis, transcription complexes were located on the yeast part of pJD14 DNA as evidenced by agarose gel electrophoresis. Southern hybridization of the pulsed RNA was restricted to a region, within the yeast DNA fragment, upstream to the initial region of the ADHI gene.

Monoclonal antibodies were raised in mouse against native RNA polymerase A from Saccharomyces cerevisiae. After screening with the spot-immunodetection technique, 14 hybridomas were selected and the antibodies produced in mice. Their specificity, analyzed by blot-immunodetection, was found to be markedly biased towards a few RNA polymerase subunits: A135 , A49 , A43 , and A14.5. A different monoclonal antibody directed against the largest subunit, A190 , was obtained by immunizing a mouse with RNA polymerase A dissociated into its subunits with SDS. Two antibodies, which probably recognized the same antigenic determinant on subunit A135 , inhibited in vitro RNA synthesis. Inhibition was prevented by preincubation of the enzyme with DNA, suggesting a role for the A135 subunit in template binding. The antibody directed against A14.5 interacted with the A14.5 kd subunit present in all three forms of the yeast nuclear RNA polymerases but did not interfere with RNA polymerase activity. These antibody probes will be useful to study subunit function in reconstituted transcription systems.

This paper describes a method for the isolation of RNA polymerase A (or I) from Saccharomyces cerevisiae which is rapid and achieves a 2,000-fold purification. The method involves mainly a batchwise adsorption of enzyme on phosphocellulose and on DEAE-cellulose and a sedimentation on glycerol gradient. The enzyme obtained can be differentiated from RNA polymerase B (or II) by a number of criteria including electrophoretic migration on polyacrylamide gel, sensitivity to α-amanitin, subunit structure, and optimal conditions for RNA synthesis. Polyacrylamide gel electrophoresis with sodium dodecyl sulfate reveals that yeast RNA polymerase A (or I) is made up of two large subunits in equimolar amount of 190,000 and 135,000 daltons and several smaller polypeptide chains, 1 (48,000), 1 (41,000), 2 (29,000), and 2 (16,000). The RNA synthesized on native calf thymus or yeast DNA is constituted of many short chains and of a small percentage (about 10%) of very long chains which represent the bulk of the RNA. Termination factor rho from Escherichia coli inhibits 50% the transcription of native DNA.

The Saccharomyces cerevisiae mutant rpo B1 produces a DNA-dependent RNA polymerase B defective in RNA synthesis in vitro. RNA polymerase B purified from the mutant is altered both structurally and functionally. The enzyme is defective in the RNA chain initiation and elongation reactions. Enzyme-DNA binding is comparatively much less affected. These enzymological defects in the mutant enzyme are enhanced at elevated ionic strengths. Purified rpo B1 RNA polymerase B is lacking B32 and B16.5 subunits. However, the low activity of the mutant enzyme cannot be accounted for only by the loss of these two polypeptides. Wild type enzyme devoid of B32 and B16.5 subunits can be obtained after a mild urea treatment. This enzyme variant, called RNA polymerase B, does not share the enzymological properties of the mutant RNA polymerase. Immunoprecipitation of the enzyme from crude extracts shows that, in the rpo B1 mutant, a normal amount of RNA polymerase B is synthesized which contains the full complement of subunits. The polypeptide chain altered by the rpo B1 mutation was identified by partial proteolysis with proteinase K in the presence of sodium dodecyl sulfate. The 35S-labeled peptide pattern generated from the B220 subunit of the mutant enzyme differs markedly from the peptide pattern of the wild type subunit. The rpo B1 mutation therefore alters the B220 subunit, suggesting a role for this subunit in RNA chain elongation and in the association of the B32 and B16.5 subunits to the RNA polymerase molecule.

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.

DNA-dependent RNA polymerases of archaebacteria not only resemble the nuclear RNA polymerases of eukaryotes rather than the eubacterial enzymes in their complex component patterns but also show striking immunochemical, i.e., structural, homology with the eukaryotic polymerases at the level of single components. Thus, eukaryotic and archaebacterial RNA polymerases are indeed of the same type, distinct from the eubacterial enzymes, which, however, are also derived from a common ancestral structure.

Studies on RNA synthesized in vitro The bulk of RNA synthesized by purified E. coli RNA polymerase on calf thymus and T7 DNA as templates, has been chromatographed on methylated serum albumin column. The elution pattern is very similar to those obtained with pulse labelled RNA from cells. The RNA's obtained in vitro are eluted, as three main fractions, namely II, III and IV, located respectively before the 16-S and 23-S RNA and after the 23-S RNA. RNA's from Fractions II and III are not aggregates made of either different RNA chains or RNA and carrier rRNA, as demonstrated by the following data: (1) Fractions II and III are present even if the carriers rRNA are omitted. (2) Thermal or mechanical degradation of the RNA lowers Fraction IV, increases Fractions II and III and induces the formation of new peaks. (3) The elongation of RNA can be limited by incorporation of cordycepin triphosphate. Under these conditions, Fraction IV disappears and Fractions II and III lose their sharpness. (4) The RNA length can be directly measured by the ratio 3H belonging to the chain nucleotides, to 32P attached to the initiator nucleotide. The evolution of this ratio through the pattern is coherent and the relation between the position of a given RNA in the elution pattern and its length, is perfectly reproducible. After 20 min synthesis, the lengths of RNA's constitutive of Fractions II, III and IV, are respectively: 1350–1500, 2700–2900 and 5000–5500 nucleotides. When the synthesis of RNA is allowed to proceed after a short pulse with a labelled nucleotide followed by a chase, the evolution of the pattern of fractions shows that the RNA of Fractions II and III are precursors of the RNA IV which are also growing. In fact, when a synchronized elongation of the RNA chains is allowed to proceed, we obtain only Fraction IV. These data, toegether with the fact that no nuclease activity has ever been detected in our preparations, show that the particular heterogeneity of the RNA is a genuine property of the material synthesized in vitro. The relative quantities of Fractions II, III and IV vary with the DNA, RNA polymerase and nucleotide concentrations. The kinetics of appearance of the different fractions and the knowledge of their respective lengths show that the average rate of RNA synthesis decreases with time of incubation. It is 90–100 nucleotides/sec during the first 30 sec and 20–27 nucleotides/sec during the first 3 min. One explains the presence of these specific intermediary fractions by the fact that the rate of synthesis is not homogeneous along the entire DNA molecule, and that the initiation of RNA chains is not instantaneous. The kinetics of the synchronized elongation of the RNA chains shows 4 plateaux during the first 3 min, even though each plateau does not represent an intermediary fraction of defined chain length. It appears nevertheless that two kinds of phenomena, one rapid, one slow, are involved in the elongation of the RNA chains.

Ribonucleases H1 and H2, purified in homogeneous form from yeast cells, specifically degrade the RNA moiety of RNA · DNA hybrids. The two enzymes are physically and functionally distinct proteins. Gel electrophoresis with sodium dodecylsulfate and gel filtration revealed that RNAase H2 is a small very basic protein composed of a single polypeptide chain of molecular weight 21000. RNAase H1, also a basic protein, is made of a polypeptide chain of molecular weight 48000 which shows a marked tendency to aggregate when subjected to polyacrylamide gel electrophoresis, gel filtration or zone sedimentation. RNAase H1 and RNAase H2 require different conditions for activity. RNAase H2 digests hybridized RNA at alkaline pH and shows an absolute requirement for divalent cations, preferably Mg2+, and for -SH groups. The enzyme liberates a mixture of oligonucleotides with 5′-phosphate termini and only a small amount of 5′-mononucleotide. It is inhibited by the - SH reagent N-ethylmaleimide. RNAase H1 is active at acid pH (around pH 6), as well as at slightly alkaline pH. It is also stimulated by divalent cations but does not need to be supplemented with -SH groups and is not sensitive to N-ethylmaleimide. With (rA)n - (dT)n as substrate, the main degradation product is 5′-AMP with some dinucleotide pApA, suggesting an exonucleolytic degradation process. The possible role of RNAases H in the cell is discussed.

Meaningful chain initiation by E. coli RNA polymerase requires the protein factor u, normally found in a tight association with the enzyme [ 1.1. On the other hand, a termination factor, named rho @) [2], causes the termination of RNA synthesis at appropriate sites on the DNA template. The functional relationship between the initiation and termination events lead us to study the molecular mechanism of chain termination and particularly the possible interaction of p with RNA polymerase and DNA. First, the binding of p to T, DNA was demonstrated and second, a complex of p and RNA polymerase was isolated by polyacrylamide gel electrophoresis.

1 In the presence of cordycepin triphosphate (3′-deoxyadenosine triphosphate), an analogue of ATP, RNA polymerase synthesizes very short RNA chains in vitro. These chains, labelled on the first nucleotide with [γ32P]GTP, are acid soluble but are retained on nitrocellulose filters. After alkaline hydrolysis, chromatography on Dowex 1 (CI−)shows that the 32P-labelled molecules behave like guanosine tetraphosphate. 2 When filtered on Sephadex G-75 the oligonucleotides synthesized are associated with a structure of high molecular weight, which is probably the complex DNA-enzyme, and remain so after ribonucleases or deoxyribonuclease treatment. 3 The number of nascent oligonucleotides measured by incorporation of [γ32P]GTP, represents only 50% of the chains initiated by guanosine in the control. This observation, together with the fact that nascent oligonucleotides are not sensitive to pancreatic or T1 ribonuclease, shows that they are involved in a structure which determines their stability. This structure could be an Enzyme-(DNA-RNA hybrid) complex at the growing point of the RNA chains. 4 The kinetics of initiation of RNA are the same whether chain propagation is prevented or not. 5 The number of RNA chains initiated per phage T7 DNA molecule has been measured with [γ32P]ATP and [γ32P]GTP. It increase linearly with the amount of RNA polymerase added although the amount of RNA made tends towards a maximum. On the other hand, the number of nascent oligonucleotides made does not increase with an excess of enzyme, which shows the existence of a finite number of initiation sites. These results are thought to indicate that initiation sites are reutilized during normal RNA synthesis. 6 The number of chains initiated by adenine and guanine per phage T7 DNA at the point where RNA synthesis, as a function of RNA polymerase concentration, departs from proportionality, is around 50.

The vitellin sack and the liver of hen's embryo contain a serinehydrolyase ( L -serine hydrolyase (adding hydrogensulfide), EC 4.2.1.22) which catalyzes the synthesis of cysteine from serine and mineral sulfide. This enzyme plays a role in a biological cycle of reactions allowing the turnover of the carbon chain of cysteine and the utilisation of sulphite for the synthesis of cysteic acid. It is shown thatvthe serinehydrolyase catalyses an activation of the β carbon of serine or cysteine, a reaction which brings an exchange between the functional group attached to the β carbon of the substrate and HO − or HS − ions. The synthesis of cysteine is a particular case of such an exchange.

After synthesis of short, nascent oligonucleotide in the presence of (32P)DNA, GTP, CTP, UTP and 3′dATP, one can excise with deoxyribonuclease a ternary complex of RNA polymerase, protected DNA and oligonucleotide, while the enzyme simply bound to the template is removed by increasing the ionic strength. This ternary complex is retained on nitrocellulose membranes. On polyacrylamide gel electrophoresis it migrates faster than RNA polymerase alone. The protected portion of the DNA is constituted of about 75 nucleotides. It might represent the sites for RNA initiation.

Compared to the bacterial situation, the transcriptional machinery of eukaryotes is remarkably complex. This complexity is evident at three levels. First, there is a multiplicity of RNA polymerases (one mitochondrial enzyme and three nuclear enzymes, the latter being designated as RNA polymerases A, B, and C or I, II, and III),* whereas there is only one enzyme in all bacteria and archebacteria thus far analyzed. Each RNA polymerase has a distinct gene specificity, with RNA polymerase A (which is nucleolar) synthesizing the large ribosomal RNA, polymerase B (which is the most sensitive to a amanitin) producing messenger RNAs, and polymerase C synthesizing small RNAs, such as 5S RNA and transfer RNAs. Second, each nuclear RNA polymerase appears to have more different polypeptide subunits than the bacterial enzyme. Third, purified eukaryotic RNA polymerases are unable to carry the accurate transcription of isolated genes in vitro, which is in contrast to bacterial holoenzymes. There appears to be no equivalent to a subunits in eukaryotic RNA polymerases, where additional proteins (transcription factors) are required to reconstitute specific transcription.

Transcription of eukaryotic transfer RNA genes involves, as a primary event, the stable binding of a protein factor to the intragenic promoter1–4. 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 genes5–8. τ, a large transcription factor purified from yeast cells4, interacts with these two control elements as shown by DNase I footprinting, exonuclease digestion, dimethyl sulphate protection experiments9,10 and by analysis of point mutations. Here we used a limited proteolysis treatment to obtain a smaller form of τ with drastically altered DNA binding properties. A protease-resistant domain interacts solely with the B block region of tRNA genes.

Yeast RNA polymerases A and C share an approximately equal to 40 kd subunit. We have identified, sequenced, and mutagenized in vitro the AC40 subunit gene. The RPC40 gene is unique in the yeast genome and is required for cell viability. This gene contains an open reading frame encoding a 37.6 kd protein having no significant homology with bacterial RNA polymerase subunits. The promoter region contains a 19 bp sequence also present in the largest subunit of RNA polymerase C. It also contains a well-conserved RPG box, a sequence found in the promoter region of many genes encoding the translational apparatus. A novel, plasmid-shuffling method was developed to isolate a large number of RPC40 ts mutants. One of these, ts4, was shown to be defective in the synthesis of RNA polymerases A and C at the restrictive temperature. In contrast, RNA polymerase B was made normally.

The specific activity of yeast RNA polymerases A or B, when complexed with P37 cofactor, compares favorably with that of E. coli RNA polymerase. The stimulation is observed only with double-stranded DNA but does not result from DNase action. The Km for nucleotide substrates and the optimal conditions of transcription are not modified. P37 stimulates RNA synthesis by ternary transcription complexes in the presence of poly(rI) which prevents reinitiations. The RNA chain length, estimated by 5' end labeling or sedimentation, is increased in the presence of P37. On the other hand, the trinucleotide synthesis, which reflects the chain initiation reaction, is not affected. Therefore, the cofactor appears to act at the elongation step of RNA synthesis.

Two forms of yeast RNA polymerase A are resolved by phosphocellulose chromatography. One of these, called RNA polymerase A, is lacking two polypeptide chains of 48,000 and 37,000 daltons. The properties of the two enzymes are compared in the present paper. RNA polymerase A transcribes d(A-T)n with a similar efficiency as the complete enzyme, but it is comparatively much less active with native DNA. The two enzymes can also be differentiated on the basis of their ionic strength and divalent cation requirements. RNA polymerase A has a particularly low activity at high salt and low Mg2+ concentrations. Thermal inactivation curves of the two enzymes are different when residual activity is assayed with native DNA. In contrast with d(A-T)n as template the apparent inactivation curves of the two enzymes are identical. The data suggest that the two dissociable polypeptide chains play an important role in transcription. The template specificity of yeast RNA polymerase B was further investigated using SV40 DNA-FI as template. RNA polymerase B is able to retain [3H]SV40 DNA-FI on nitrocellulose filters but the enzyme-DNA complex is very unstable. The observation that RNA polymerase B can transcribe to some extent a supercoiled DNA but not a linear double stranded template supports the hypothesis that the enzyme needs some unpaired DNA structure to initiate transcription.

In exponentially growing cells, RNA polymerase B is exclusively form BI enzyme with several phosphorylated subunits: B220, B23 and possibly B44.5. In RNA polymerase A an average of fifteen phosphate groups are distributed on the five phosphorylated subunits: A190 (6), A43 (4), A34.5 (2), A23 (1—2) and A19 (1—2). Phosphorylation of enzyme A by a yeast protein kinase in vitro adds less than 1 mol phosphate/mol enzyme but occurs essentially at the physiological sites, as shown by a comparison of the peptide patterns obtained by limited proteolysis of subunits 32P-labelled in vivo and in vitro. No evidence was found in favour of a modulation of RNA polymerase activity in vitro or in vivo via phosphorylation.

Yeast transcription factor tau interacts with the intragenic promoter of tRNA genes, binding to both the A and B block elements. Affinity-purified tau factor and tau-tDNA complexes were examined by scanning transmission electron microscopy to analyze the structural features of free and DNA bound factor. The free factor appeared as two tightly associated globular domains of roughly similar size (10 nm in diameter) and mass (approximately 300 kd). A combination of these two domains results in a mass for the factor of 510-670 kd. When tau was allowed to interact with recombinant tRNA(3Leu) genes with variable A block-B block spacing, different structures were observed. With short genes, the two globular domains were not resolved and tau appeared as a large particle covering the A and B block region. On the other hand, with genes having a larger A-B distance (53 or 74 bp), mostly dumb-bell-shaped complexes were formed with individualized factor domains bound separately to the A and B blocks. A smaller proportion of the complexes appeared to consist of a large particle bound at only one site, essentially on the B block. Mapping of the binding domains in the DNA showed a good correlation with the respective positions of the A and B promoter elements. Factor binding did not induce a noticeable DNA bending, although with extended genes apparent DNA shortening and cases of DNA looping were observed. Upon cleavage of the tRNA(3Leu) gene between the A and B blocks after or prior to complex formation, the two factor domains remained attached to the same DNA fragment (mostly the B-DNA fragment). In addition, images of protein-linked, reconstituted full-length genes were also observed. These different conformational states of the tau-tDNA complexes probably reflect the dynamic aspect of the interaction of the factor with its DNA target.

Summary A hybrid plasmid, containing yeast ribosomal DNA, was transcribed by purified yeast RNA polymerase A in the presence of the stimulation factor P37. The transcription was not random. The yeast sequences were copied highly asymetrically, as shown by competition hybridization with yeast ribosomal RNA. Using the same DNA, truncated by different restriction enzymes, it appeared, however, that the in vitro preferential initiation region was located upstream of the presumptive in vivo initiation site for the 37S pre-rRNA.

There is a natural variation in the molecular structure of RNA polymerase A isolated from several genetically distant yeast species, Saccharomyces cerevisiae, Saccharomyces douglasii, Schizosaccharomyces pombe, and Candida tropicalis. Several biochemical criteria were used to identify their homologous polypeptide components. Based on these correlations, the minimal subunit composition of S. cerevisiae (and Saccharomyces carlsbergensis) RNA polymerase A was tentatively defined as A190, A135, A40, A27, A23, A19, and A14.5. Without the two Saccharomyces species, S. cerevisiae and S. douglasii, 7 of 13 polypeptides of enzyme A(A49, A43, A40, A34.5, A19, A14.5, and A14) differ slightly in molecular weight and can be resolved by electrophoresis on polyacrylamide gel. The RNA polymerase A isolated from the diploid interspecific hybrid contains all the polypeptides characteristic of the two parents. One meiotic segregant had a hybrid RNA polymerase A with five of the polymorphic polypeptides (A49, A43, A19, A14.5, and A14) coming from S. douglasii and two (A40 and A34.5) from S. cerevisiae. In three successive backcrosses with S. cerevisiae, all the genes for S. douglasii polypeptides were shown to recombine although parental ditype tetrads predominated in the four four-spored asci examined. Thus, the genes for the seven polymorphic polypeptides are not clustered: they lie on at least three different chromosomes.

A latent RNAase activity stimulated by nucleoside triphosphates has been isolated from a yeast chromatin extract, by filtration on Sepharose 6B and hydroxyapatite chromatography. The RNAase was separated from a thermolabile proteic inhibitor on phosphocellulose. When separated from the inhibitor, the RNAase hydrolyses RNA to 5′-mononucleotides. Its activity is retained in the presence of EDTA, and 50% inhibited by 1 mM ATP or CTP. The RNAase is inhibited by the thermolabile component only in the presence of divalent cations. The activity is recovered upon addition of 0.01 mM ATP to the mixture. The K m for ATP is 10 μM. ATP can be replaced by other ribo- or deoxyribonucleoside triphosphates with varying efficiency but not by ADP, AMP or cAMP. These results suggest multiple interactions between the RNAase, a regulatory component, divalent cations and nucleoside triphosphates.

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 tRNA1–3. 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 recognition6. By analogy with the 5S RNA system, where a transcription factor required for 5S DNA expression was shown to interact both with 5S RNA7,8 and with the noncoding strand of the 5S gene9, 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.

Two ribonuclease H activities have been found in yeast RNA polymerase A. The nuclease activities comigrated with subunits A49 (Mr = 49,000) and A40 (Mr = 40,000), after electrophoresis in a sodium dodecyl sulfate polyacrylamide gel containing [32P](rG)n . (dC)n as substrate. Both activities were also found, among other nucleases, in a high salt chromatin extract. Several lines of evidence suggest that the chromatin RNase H of 49,000 daltons (RNase H49) is the same protein as subunit A49. They co-migrate on sodium dodecyl sulfate-gel electrophoresis, have the same chromatographic properties, and dissociate simultaneously from RNA polymerase A. Fractions containing RNase H49 stimulate RNA synthesis by RNA polymerase A* lacking A49 and A34.5 subunits. Finally, limited proteolysis of the protein band having RNase H49 activity yields the characteristic fingerprint of the A49 subunit. This subunit, therefore, exists in two states: bound to chromatin and associated with RNA polymerase A. On the other hand, it is not yet clear whether the RNase H activity of 40,000 daltons, associated with RNA polymerase A, is due to the A40 subunit or whether it represents a trace contamination by a very active nuclease tightly bound to the enzyme.

A basic 37,000-dalton protein (P37), purified from yeast cells, interacts with yeast RNA polymerase B and drastically increases its specific activity. A complex of P37 and RNA polymerase can be isolated by sedimentation through a glycerol gradient. The complex is dissociated at the ionic strength of 0.9. The preferential binding of P37 with RNA polymerase form BI (with the unproteolyzed B220 subunit) was visualized by polyacrylamide gel electrophoresis under nondenaturing conditions. Kinetic analysis of the RNA polymerase cofactor interaction indicated that the dissociation constant for the complex is 5 X 10(-8) M.

Publisher Summary This chapter describes the eukaryotic RNA polymerases. The RNA polymerase A was subjected to mild urea treatment. The enzyme was treated with 4M urea that resulted in 80% inactivation. Then the partially dissociated enzyme was subjected to chromatography on phosphocellulose and the fractions were analyzed for polypeptide pattern, by SDS gel electrophoresis, and for specific activity. Several acidic polypeptides were found in the flow-through. No activity was recovered in this fraction. The salt gradient then eluted a complex of which retained only 8% of original specific activity. Following this, some enzyme was eluted, containing, in addition to the above subunits, A49 and A34.5, with 35% of activity. The two basic polypeptides A43 and A34.5 were eluted together with different amounts of various other subunits. This fraction was inactive but stimulated two fold fraction b but not fraction c. Partial dissociation of RNA polymerase B with urea leads to an amplification of the spontaneous dissociation of two polypeptides.

Specific antibodies directed against each polypeptide component of yeast RNA polymerases A or B were prepared and their affinity spectrum determined by protein blot immunodetection. The majority of enzyme A or B subunits were specifically recognized by their respective antiserum. A direct correspondence was established between the polypeptides immunologically related in the three forms of RNA polymerases A, B, and C by reacting the different antibodies with enzymes subunits transferred to a nitrocellulose membrane. Subunit-specific antibodies and antibodies to native enzymes A and B were used to probe the activity of RNA polymerases A, B, and C. Based on DNA protection experiments, the largest subunit of enzymes A and B as well as the common subunit ABC23 appear to be involved in DNA binding.

A method was devised for directing RNA polymerase on a single promoter site on T7 DNA. Initiation complexes were formed on each of the three main promoter sites using one dinucleotide plus one nucleoside triphosphate. The ternary initiation complexes are resistant to rifampicin action, to inhibition by (rI)n at 0 °C and are stable at high salt concentrations. A minimum of a trinucleotide is required to form a stable ternary complex. To determine which promoter site was selected by RNA polymerase during initiation, the (rI)n-resistant RNA was digested by RNAse III to generate three characteristic initiator RNA fragments, resolved by gel electrophoresis. The three major promoter sites could be selected individually by using different primer and substrate combinations: ApC + ATP selected promoter A3, CpG + CTP selected A2 and CpC + ATP specified preferentially A1. A number of primer-substrate combinations specified each site at low salt concentration but the substrate requirement became very stringent at high salt concentration, suggesting that the postulated local opening of the promoter site could be more or less extensive, depending on the ionic strength. The minimum opening observed at high salt concentration corresponded to the insertion of a leader trinucleotide sequence. The promoter region melted by RNA polymerase at low salt concentration was (G + C)-rich and corresponded to about 9 to 11 base pairs. Sequences of the melting recognition regions were tentatively inferred from the results.

The factor TUF interacts specifically with RPG or HOMOL1 sequences, which are present upstream of many genes coding for the yeast translational apparatus. Here we present evidence that the RPG and HOMOL1 motifs are variants of a consensus UASrpg (upstream activating sequence) recognized by the same factor. Factor TUF was identified by using two highly selective methods. The DNA-protein complex was isolated by pore-limit electrophoresis in polyacrylamide gradient gels and found to contain a single polypeptide of 150 kDa. In a two-step protein-blotting/nuclease-protection ("footprinting") procedure, the same 150-kDa polypeptide blotted on nitrocellulose exhibited the same specific DNA-binding properties as TUF factor. A 50-kDa DNA-binding domain of TUF was isolated by selective proteolysis. This suggests a bipolarization of the TUF protein, with distinct functional domains.

Two hundred strains of Saccharomyces cerevisiae temperature sensitive for RNA synthesis were selected and screened in crude extracts for DNA-dependent RNA polymerase activities. One strain was isolated which had only residual in vitro RNA polymerase B activity. In normal growth conditions total RNA, poly A+ RNA and protein synthesis were indistinguishable from those of the wild type strain at 23 degrees C and after shift to 37 degrees C. A temperature sensitive phenotype was detected only when rpoB containing strains were grown in adverse conditions. The mutant character showed mendelian segregation and was coexpressed with the wild type character in heterozygous diploids. Residual enzyme activity was characterised in crude extracts using synthetic polymers and natural templates in different ionic conditions.

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.

The isolation and characterization of temperature-sensitive mutations in RNA polymerase I from Saccharomyces cerevisiae are described. A plasmid carrying RPA190, the gene encoding the largest subunit of the enzyme, was subjected to in vitro mutagenesis with hydroxylamine. Using a plasmid shuffle screening system, five different plasmids were isolated which conferred a temperature-sensitive phenotype in haploid yeast strains carrying the disrupted chromosomal RPA190 gene. These temperature-sensitive alleles were transferred to the chromosomal RPA190 locus for mapping and physiology experiments. Accumulation of RNA was found to be defective in all mutant strains at the nonpermissive temperature. In addition, analysis of pulse-labeled RNA from two mutant strains at 37 degrees C showed that the transcription of rRNA genes was decreased, while that of 5S RNA was relatively unaffected. RNA polymerase I was partially purified from several of the mutant strains grown at the nonpermissive temperature and was shown to be deficient when assayed in vitro. Fine-structure mapping and sequencing of the mutant alleles demonstrated that all five mutations were unique. The rpa190-1 and rpa190-5 mutations are tightly clustered in region I (S.S. Broyles and B. Moss, Proc. Natl. Acad. Sci. USA 83:3141-3145, 1986), the putative zinc-binding region that is common to all eucaryotic RNA polymerase large subunits. The rpa190-3 mutation is located between regions III and IV, and a strain carrying it behaves as a mutant that is defective in the synthesis of the enzyme. This mutation lies within a previously unidentified segment of highly conserved amino acid sequence homology that is shared among the largest subunits of eucaryotic nuclear RNA polymerases. Another temperature-sensitive mutation, rpa190-2, creates a UGA nonsense codon.

Special care to prevent proteolysis during yeast RNA polymerase B purification leads to the appearance of two forms of enzymes, BI and BII, with different molecular weight (465 000) and 435 000, respectively). The two forms of enzyme can be separated by ion-exchange chromatography or polyacrylamide gel electrophoresis. Their subunit structures were compared by sodium dodecylsulfate gel electrophoresis, the only observed difference between the two enzymes is in the molecular weight of the heaviest subunit which is 220 000 for enzyme BI and 180 000 for enzyme BII. Otherwise, the two enzymes have seven common subunits of molecular weights 150 000, 45 000, 26 000, 22 500, 14 500, 12 500 and 9000. Two additional polypeptide chains of 32 000 and 16 500 Mr are dissociated from the enzyme upon polyacrylamide gel electrophoresis or DEAE Sephadex chromatography. The largest subunit of enzyme BI (Mr 220 000) can be specifically cleaved in vitro by a yeast protease extract, generating a polypeptide chain indistinguishable from the largest subunit of enzyme BII. This proteolytic cleavage of enzyme BI in vitro is inhibited by phenylmethylsulfonyl fluoride and does not significantly change the activity of the enzyme with single-stranded or double-stranded DNA as template. The precursor-product relationship of the different forms of class B RNA polymerases in eukaryotic cells is discussed.

With the object of isolating yeast mutants altered in DNA-dependent RNA polymerases, a specific assay for the various classes of RNA polymerases was developed, using crude protein extracts as the source of enzyme. Yeast extracts incorporated AMP as well as UMP and CMP residues into acid-precipitable material in template-independent reactions which obscured DNA-dependent RNA polymerase activities. In contrast, GMP incorporation was exclusively template-dependent. Therefore cytidinerich templates were selected. A simple and sensitive assay for RNA polymerase B was based on its ability to use the ribohomopolymer (rC)n as template and its exclusive requirement for Mn2+ as activator cation. The specificity of the assay was further shown by the fact that (rG)n synthesis under these conditions was totally inhibited by 50 μg/ml of α-amanitin, a sensitivity ascribed only to RNA polymerase B. The other two forms of enzymes, RNA polymerases A and C, were assayed with d(I-C)n as template in the presence of Mg2+ as activator cation, under the correct ionic conditions. At low ionic strength and in the presence of Mg2+, d(I-C)n-directed synthesis of r(C-G)n was catalyzed by enzymes A and C. In contrast, at high ionic strength or in the presence of 2 mg/ml of α-amanitin, the activity of RNA polymerase A was inhibited (∼ 50%). Thus the activities of A and C enzymes could be calculated by the difference of activities measured on low salt and high salt. It was found that RNA polymerases A and C each contributed to about 50% of the r(G-C)n synthesis. Several parameters influencing the assay were investigated. In particular, RNA polymerase activities were found to be independent of cell growth state. In order to use these methods for the rapid screening of a large number of clones, the processing of the assays was modified to permit a rapid comparison by visual tests.