Similar upstream regulatory elements of genes that encode the two largest subunits of RNA polymerase II in Saccharomyces cerevisiae.

We have determined the location of cis-acting elements that are important for the expression of RPO21 and RPO22, genes that encode the two largest subunits of RNA polymerase II (RNAPII) in Saccharomyces cerevisiae. A series of 5'-end deletions and nucleotide substitutions in the upstream regions of RPO21 and RPO22 were tested for their effect on the expression of lacZ fusions of these genes. Deletion of sequences from -723 to -693 in RPO21, which disrupted two Reb1p-binding sites and an Abf1p-binding site, resulted in a 10-fold decrease in expression. A T-rich region downstream of these sites was also important for expression. Deletion of sequences from -437 to -392 in the RPO22-upstream, which resulted in a 30-fold decrease in expression, indicated that the Reb1p- and Abf1p-binding sites in this region were important for RPO22 expression, as was a T-rich sequence immediately downstream of these sites. The RPO21 and RPO22 upstream regions were capable of interacting in vitro (gel-mobility-shift assays) with Reb1p and Abf1p. The similarities in the type and organization of elements in the upstream regions of RPO21 and RPO22 suggest that expression of these genes may be regulated coordinately.     

Intracellular contents and assembly states of all 12 subunits of the RNA polymerase II in the fission yeast Schizosaccharomyces pombe.

The RNA polymerase II (Pol II) of the fission yeast Schizosaccharomyces pombe is composed of 12 different polypeptides, Rpb1 to Rpb12, of which five, Rpb5, Rpb6, Rpb8, Rpb10 and Rpb12, are shared among three forms of the RNA polymerase. To get an insight into the control of synthesis and assembly of individual subunits, we have measured the intracellular concentrations of all 12 subunits in S. pombe by quantitative immunoblotting. Results indicate that the levels are low for the three large subunits, Rpb1, Rpb2 and Rpb3, which are the homologues of beta', beta and alpha subunits, respectively, of prokaryotic RNA polymerase. On the other hand, the levels of small-sized subunits were between 2- to 15-fold higher than these three core subunits. The levels of the five common subunits shared among RNA polymerases I, II and III are about 10 times greater than those of the Pol II-specific core subunits. The assembly state of the Rpb proteins was analyzed by glycerol gradient centrifugation of S. pombe whole cell extracts. The three core subunits are mostly assembled in Pol II, but some of the small subunits were detected in the slowly sedimenting fractions, indicating that at least some of the excess Rpb proteins exist in unassembled forms. Based on the intracellular concentration of the least abundant Rpb3 subunit, the total number of Pol II in a growing S. pombe cell was estimated to be about 10,000 molecules. The intracellular distribution of some Pol II subunits was also analyzed by microscopic observation of the green fluorescent protein (GFP)-fused Rpb proteins. In agreement with the biochemical analysis, the GFP-Rpb1 and GFP-Rpb3 fusions were present in the nuclei but the GFP-Rpb4 was detected in the cytoplasm as well as the nuclei.     

Localization of the yeast RNA polymerase I-specific subunits

The spatial distribution of four subunits specifically associated to the yeast DNA-dependent RNA polymerase I (RNA pol I) was studied by electron microscopy. A structural model of the native enzyme was determined by cryo-electron microscopy from isolated molecules and was compared with the atomic structure of RNA pol II Delta 4/7, which lacks the specific polypeptides. The two models were aligned and a difference map revealed four additional protein densities present in RNA pol I, which were characterized by immunolabelling. A protruding protein density named stalk was found to contain the RNA pol I-specific subunits A43 and A14. The docking with the atomic structure showed that the stalk protruded from the structure at the same site as the C-terminal domain (CTD) of the largest subunit of RNA pol II. Subunit A49 was placed on top of the clamp whereas subunit A34.5 bound at the entrance of the DNA binding cleft, where it could contact the downstream DNA. The location of the RNA pol I-specific subunits is correlated with their biological activity.     

A protein kinase from wheat germ that phosphorylates the largest subunit of RNA polymerase II.

A protein kinase from wheat germ that phosphorylates the largest subunit of RNA polymerase IIA has been partially purified and characterized. The kinase has a native molecular weight of about 200 kilodaltons. This kinase utilizes Mg2+ and ATP and transfers about 20 phosphates to the heptapeptide repeats Pro-Thr-Ser-Pro-Ser-Tyr-Ser in the carboxyl-terminal domain of the 220-kilodalton subunit of soybean RNA polymerase II. This phosphorylation results in a mobility shift of the 220-kilodalton subunits of a variety of eukaryotic RNA polymerases to polypeptides ranging in size from greater than 220 kilodaltons to 240 kilodaltons on sodium dodecyl sulfate-polyacrylamide gels. The phosphorylation is highly specific to the heptapeptide repeats since a degraded subunit polypeptide of 180 kilodaltons that lacks the heptapeptide repeats is poorly phosphorylated. Synthetic heptapeptide repeat multimers inhibit the phosphorylation of the 220-kilodalton subunit.     

Mutations in the rpmBG operon of Escherichia coli that affect ribosome assembly.

The rpmBG operon of Escherichia coli codes for ribosomal proteins L28 and L33. Two strains with mutations in the operon are AM81, whose ribosomes lack protein L28, and AM90, whose ribosomes are without protein L33. Neither strain showed major defects in ribosome assembly. However, when the mutations were transferred to other strains of E. coli, ribosome synthesis was greatly perturbed and precursor ribonucleoproteins accumulated. In the new backgrounds, the mutation in rpmB was complemented by synthesis of protein L28 from a plasmid; the rpmG mutation was not complemented by protein L33 because synthesis of protein L28 from the upstream rpmB gene was also greatly reduced. The results suggest that protein L33, in contrast to protein L28, has at best a minor role in ribosome assembly and function.     

Arabidopsis thaliana RNA polymerase II subunits related to yeast and human RPB5

Arabidopsis thaliana contains at least four genes that are predicted to encode polypeptides related to the RPB5 subunit found in yeast and human RNA polymerase II. This subunit has been shown to be the largest subunit common to yeast RNA polymerases I, II, and III (RPABC27). More than one of these genes is expressed in Arabidopsis suspension culture cells, but only one of the encoded polypeptides is found in purified RNA polymerases II and III. This polypeptide has a predicted pI of 9.6, matches 14 of 16 amino acids in the amino terminus of cauliflower RPB5 that was microsequenced, and shows 42 and 53% amino acid sequence identity with the yeast and human RPB5 subunits, respectively.     

Protein kinases from Aspergillus nidulans that phosphorylate the carboxyl-terminal domain of the largest subunit of RNA polymerase II.

Three serine kinases which phosphorylate the CTD of RNA polymerase II have been identified in Aspergillus nidulans. The kinases (KI, KII, KIII) were identified using a synthetic peptide containing four copies of the CTD consensus heptamer repeat, and differ in chromatographic behavior, and apparent molecular mass (KI approximately 60kDa; KII approximately 82kDa; KIII approximately 43 kDa). KIII utilized, in addition to peptide, histone H1 as substrate, whereas casein was not phosphorylated by any of the three kinases. The kinases appear to be unrelated to the p34cdc2 kinase, as judged by Western blot analysis and the position of serine phosphorylation of the synthetic CTD peptide. KI was highly purified and renaturation experiments have shown that it consists of a single polypeptide of 57 kDa. KI also phosphorylated RNA polymerase II associated in a preinitiation complex.     

Genetic exploration of interactive domains in RNA polymerase II subunits.

The two large subunits of RNA polymerase II, RPB1 and RPB2, contain regions of extensive homology to the two large subunits of Escherichia coli RNA polymerase. These homologous regions may represent separate protein domains with unique functions. We investigated whether suppressor genetics could provide evidence for interactions between specific segments of RPB1 and RPB2 in Saccharomyces cerevisiae. A plasmid shuffle method was used to screen thoroughly for mutations in RPB2 that suppress a temperature-sensitive mutation, rpb1-1, which is located in region H of RPB1. All six RPB2 mutations that suppress rpb1-1 were clustered in region I of RPB2. The location of these mutations and the observation that they were allele specific for suppression of rpb1-1 suggests an interaction between region H of RPB1 and region I of RPB2. A similar experiment was done to isolate and map mutations in RPB1 that suppress a temperature-sensitive mutation, rpb2-2, which occurs in region I of RPB2. These suppressor mutations were not clustered in a particular region. Thus, fine structure suppressor genetics can provide evidence for interactions between specific segments of two proteins, but the results of this type of analysis can depend on the conditional mutation to be suppressed.     

Amanitin-resistant RNA polymerase II mutations are in the enzyme's largest subunit

A fragment of the Drosophila melanogaster RpIIC4 locus, which encodes the RNA polymerase II subunit that determines amanitin sensitivity, was inserted into a bacterial plasmid cloning vehicle useful for over-production of hybrid proteins. Two plasmid constructions encoded hybrid proteins that reacted with antibodies against D. melanogaster RNA polymerase II. Use of subunit-specific antibodies indicated that these hybrid proteins displayed antigenic determinants unique to the largest polypeptide (215 kDa) of the enzyme. This RpII locus, the site at which mutations to amanitin-resistance occur, must therefore encode the largest polymerase II subunit.