Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have examined the translational coupling between the first two genes in the S10 ribosomal protein operon. We isolated mutations blocking the translation of the first gene of the operon, coding for S10, and monitored their effects on translation of the downstream gene, coding for L3. All of the mutations inhibiting S10 synthesis also affected the synthesis of L3. However, these experiments were complicated by decreased mRNA synthesis resulting from transcription polarity, which we could only partially eliminate by using a rho-100 strain. To completely eliminate the problem of transcription polarity and obtain a more accurate measurement of the coupling, we replaced the natural S10 promoter with a promoter used by the bacteriophage T7 RNA polymerase. As expected, the T7 RNA polymerase was not subject to transcription polarity. Using this system, we were able to show that a complete abolishment of S10 translation resulted in an 80% inhibition of L3 synthesis. Other experiments show that the synthesis of L3 goes up as a function of increasing S10 synthesis, but the translational coupling does not assure strictly proportional output from the two genes.
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PMID:Translational coupling of the two proximal genes in the S10 ribosomal protein operon of Escherichia coli. 265 12

Bacteriophage lambda N gene product acts to modify host RNA polymerase allowing the formation of a termination-resistant transcription apparatus. Previous studies have demonstrated that the nusE71 mutation that has altered the ribosomal protein S10 prevents N action in vivo. Using a coupled transcription-translation system, we demonstrate here that purified S10 protein as well as the 30S ribosomal subunit is sufficient to restore N activity in the nusE mutant extract, allowing antitermination of Rho-dependent and Rho-independent terminators. This provides direct biochemical evidence that the S10 protein itself is one of the cellular components necessary for the formation of an antitermination apparatus.
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PMID:Evidence that ribosomal protein S10 itself is a cellular component necessary for transcription antitermination by phage lambda N protein. 298 61

The nucleotide sequence (25,320 base-pairs) of a part of the large single-copy region of chloroplast DNA from the liverwort Marchantia polymorpha was determined. This region encodes putative genes for four tRNAs, isoleucine tRNA(CAU), arginine tRNA(CCG), proline tRNA(UGG) and tryptophan tRNA(CCA); eight photosynthetic polypeptides, the large subunit of ribulose bisphosphate carboxylase/oxygenase (rbcL), 51,000 Mr photosystem II chlorophyll alpha apoprotein (psbB), apocytochrome b-559 polypeptides (psbE and psbF), 10,000 Mr phosphoprotein (psbH), cytochrome f preprotein (petA), cytochrome b6 polypeptide (petB), and cytochrome b6/f complex subunit 4 polypeptide (petD); 13 ribosomal proteins (L2, L14, L16, L20, L22, L23, L33, S3, S8, S11, S12, S18 and S19); initiation factor 1 (infA); ribosome-associating polypeptide (secX); and alpha subunit of RNA polymerase (rpoA). Functionally related genes were located in several clusters in this region of the genome. There were two ribosomal protein gene clusters: rpl23-rpl2-rps19-rpl22-rps3-rpl16-+ ++rpl14-rps8-infA-secX-rps11-rpoA, with a gene arrangement similar to that of the Escherichia coli S10-spc-alpha operons, and the rps12'-rpl20-rps18-rpl33 cluster. There were gene clusters encoding photosynthesis components such as the psbB-psbH-petB-petD and the psbE-psbF clusters. Thirteen open reading frames, ranging in length from 31 to 434 amino acid residues, remain to be identified.
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PMID:Structure and organization of Marchantia polymorpha chloroplast genome. III. Gene organization of the large single copy region from rbcL to trnI(CAU). 319 36

In vitro synthesis of a number of E. coli 30S ribosomal proteins has been demonstrated in a cell-free system consisting of ribosomes, initiation factors, RNA polymerase, a fraction containing soluble enzymes and factors, and E. coli DNA. DNA-dependent synthesis of the following 30S proteins has been demonstrated: S4, S5, S7, S8, S9, S10, S13, S14, S16, S19, and S20.
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PMID:In vitro synthesis of ribosomal proteins directed by Escherichia coli DNA. 420 93

We have probed regions of the S10 leader RNA to determine their role in L4-mediated, NusA-dependent attenuation control of the S10 ribosomal protein operon. Using genetic and "antisense" oligonucleotide competition approaches, we were able to distinguish between the determinants necessary for intrinsic (NusA-independent) pausing by RNA polymerase at the S10 attenuation site, for NusA-dependent enhancement of pausing, and for L4 stabilization of the paused ternary complex. The upper stem-loop structure in the attenuator hairpin is the major determinant for the NusA-dependent pause, while the sequence at the site of pausing is important for RNA polymerase to pause in the absence of NusA. The determinants for L4 stabilization of the paused complex include the hairpin immediately upstream of the attenuator hairpin as well as the ascending side of the attenuator structure. In conclusion, our results suggest that there are three distinct pausing activities by RNA polymerase during its transcription of the S10 leader, with three corresponding signals in the S10 leader.
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PMID:RNA determinants required for L4-mediated attenuation control of the S10 r-protein operon of Escherichia coli. 753 Dec 46

The stable association of the N gene transcriptional antiterminator protein of bacteriophage lambda with transcribing RNA polymerase requires a nut site (boxA+boxB) in the nascent transcript and the Escherichia coli factors NusA, NusB, NusG, and ribosomal protein S10. We have used electrophoretic mobility shift assays to analyze the assembly of N protein, the E. coli factors, and RNA polymerase onto the nut site RNA in the absence of a DNA template. We show that N binds boxB RNA and that subsequent association of NusA with the N-nut site complex is facilitated by both boxA and boxB. In the presence of N, NusA, and RNA polymerase the nut site assembles ribonucleoprotein complexes containing NusB, NusG, and S10. The effects on assembly of mutations in boxA, boxB, NusA, and RNA polymerase define multiple weak protein-protein and protein-RNA interactions (e.g., NusB with NusG; NusA with boxB; NusA, NusB, and NusG with boxA) that contribute to the overall stability of the complex. Interaction of each component of the complex with two or more other components can explain the many observed cooperative binding associations in the DNA-independent assembly of a stable antitermination complex on RNA polymerase.
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PMID:A protein-RNA interaction network facilitates the template-independent cooperative assembly on RNA polymerase of a stable antitermination complex containing the lambda N protein. 759 Feb 57

The boxA sequences of the E. coli ribosomal RNA (rrn) operons are sufficient to cause RNA polymerase to read through Rho-dependent transcriptional terminators. We show that a complex of the transcription antitermination factors NusB and ribosomal protein S10 interacts specifically with boxA RNA. Neither NusB nor S10 binds boxA RNA on its own, and neither NusA nor NusG affects the interaction of the NusB-S10 complex with boxA RNA. Mutations in boxA that impair its antitermination activity compromise its interaction with NusB and S10, suggesting that ribosomal protein S10 regulates the synthesis of ribosomal RNA in bacteria. RNA containing the closely related boxA sequence from the bacteriophage lambda nutR site is not stably bound by NusB and S10. This probably explains why antitermination in phage lambda depends on the phage lambda N protein and the boxB component of the nut site, in addition to boxA.
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PMID:Recognition of boxA antiterminator RNA by the E. coli antitermination factors NusB and ribosomal protein S10. 767 81

The transcription of the 11 gene S10 operon of Escherichia coli is autogenously regulated by one of the operon's products, ribosomal protein L4. This protein stimulates termination of transcription in vivo at a specific site within the S10 leader. The in vivo effect can be reproduced in a purified transcription system but requires an additional factor, NusA. Our earlier in vitro studies showed that NusA is required for RNA polymerase pausing at the termination site; such paused complexes are further stabilized by L4, which presumably accounts for L4's stimulation of termination in vivo. Here we show that NusA is not absolutely required for RNA polymerase to recognize the attenuation site: at low (5 microM) UTP concentration, RNA polymerase pauses at the site, although the paused transcription complex formed in the absence of NusA can be further stabilized by subsequent addition of the protein. Furthermore, RNA polymerase pausing at the attenuation site is not sufficient for the L4 effect, since L4 cannot stabilize a transcription complex paused at the attenuation site in the absence of NusA. We have been able to isolate paused complexes formed without NusA and/or L4; such complexes are active upon re-addition of NTPs, and respond as expected to the addition of L4 or NusA. Our experiments are consistent with the notion that L4 is a stable component of a paused transcription complex.
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PMID:Role of NusA in L4-mediated attenuation control of the S10 r-protein operon of Escherichia coli. 784 21

URP2 was cloned as a multicopy suppressor of several temperature-sensitive mutations defective in RNA polymerase III-dependent transcription, but without effect on mutations affecting RNA polymerase I or II. This single-copy gene encodes a hydrophilic polypeptide of 121 amino acid residues with a predicted molecular mass of 13.9 kDa and a basic isoelectric point of 9.7. URP2 is a highly expressed gene, judging from its abundant messenger RNA and strong codon bias. The Urp2p protein is essential for cell growth, as shown by the lethal phenotype of the urp2::HIS3 null allele. Given its striking similarity to the S20 ribosomal polypeptide of rat (55% identical residues), Urp2p is in all likelihood the yeast form of this polypeptide. Both proteins are significantly related to S10, a component of the small ribosomal subunit of Escherichia coli that is known to operate as a transcriptional elongation factor. The latter observation suggests that the suppressor effect of URP2 may be due to a direct involvement of Urp2p in RNA polymerase III-dependent transcription. Alternatively, the overexpression of Urp2p could bypass a partial preribosomal RNA processing defect associated with RNA polymerase III mutants. URP2 was assigned to the left arm of chromosome VIII, and maps between DUR3 and YLF1. The latter gene product has homology to the E. coli gtp1 gene product, and may define a new family of putative GTP-binding proteins.
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PMID:Suppression of yeast RNA polymerase III mutations by the URP2 gene encoding a protein homologous to the mammalian ribosomal protein S20. 802 36

The ability of phosphorothioate (POS) oligonucleotides to recognise and bind to homopurine-homopyrimidine DNA double-stranded sites via triple helix formation has been investigated. It has been found that the homologous pyrimidine POS sequences Y11-Si (i = 0, 1,2,3,4,10), which have been obtained by an increasing sulphur substitution in the sugar-phosphate backbone of d(CTTCCTCCTCT) (Y11), and the target hairpin duplex d(GAAGGAGGAGA-T4-TCTCCTCCTTC) (h26) can form stable triple helices, as indicated by PAGE, CD and UV melting experiments. The thermal stability of the triple helices depends on the number of POS linkages in the third Y11 strand, varying from 48 degrees C (Y11, with only phosphate groups, PO2) to 31 degrees C (Y11-S10 containing exclusively thioate groups). On average, a Tm depression of about 2 degrees C per POS linkage introduced in Y11 was observed. CD data indicate that the sulphurization of the third strand results in minimal changes of triple-stranded structures. The energetics of the triplex-to-hairpin plus single-strand transition has been determined by van't Hoff analyses of the melting curves. In free energy terms, the POS triplexes h26.Y11-Si are less stable than the normal PO2 h26.Y11 triplex by values between 2.7 and 5.4 kcal/mol, depending on the number of POS linkages contained in the third strand. Phosphorothioate oligonucleotides being resistant towards several nucleases offer an interesting choice as gene blockers in antisense strategy. Thus, their ability to inhibit transcription via triple helix formation has been examined in vitro. We found that triplex-forming POS oligonucleotides of 20 bases in length (with a cytosine contents of 45%), containing either 10% or 26% thioate groups, strongly repress the transcription activity of the bacteriophage T7 RNA polymerase at pH 6.9, when used in excess compared to the target (mol oligo/mol template = 125). The here reported data are useful for designing phosphorothioate oligonucleotides targeted to genomic DNA in antigene strategy.
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PMID:Pyrimidine phosphorothioate oligonucleotides form triple-stranded helices and promote transcription inhibition. 807 67


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