scholarly journals Purines are required at the 5' ends of newly initiated RNAs for optimal RNA polymerase III gene expression.

1996 ◽  
Vol 16 (10) ◽  
pp. 5801-5810 ◽  
Author(s):  
G N Zecherle ◽  
S Whelen ◽  
B D Hall
Keyword(s):  
Rna Polymerase ◽  
Ternary Complex ◽  
Rna Polymerase Iii ◽  
Phenotypic Change ◽  
Start Site ◽  
Transcription Start ◽  
Polymerase Iii ◽  
Nascent Transcripts ◽  
Mutant Genes

We have made specific alterations in the CAACAA element at the transcription start site of a Saccharomyces cerevisiae suppressor tRNA gene. The mutant genes were tested for their ability to suppress the ochre nonsense alleles ade2-1, lys4-1, and met4-1. Many of the mutants showed either no phenotypic change or a weak loss of suppression relative to that of SUP4-o. A 2-bp change, CTCCAA, which alters bases encoding the +1 and +2 nucleotides of pre-tRNA Tyr, had a strong deleterious effect in vivo, as did the more extensive change CTCCTC. In contrast, mutant genes bearing each of the possible single changes at nucleotide +1 retained normal suppression levels. The transcription start point could be shifted in a limited fashion in response to the specific sequences encountered by RNA polymerase III at the start site. ATP was preferentially utilized as the 5' nucleotide in the growing RNA chain, while with start site sequences that precluded utilization of a purine, CTP was greatly preferred to UTP as the +1 nucleotide. Short oligopyrimidine RNAs formed on the CTCCTC allele could be repositioned in the active center of the newly formed ternary complex. Early postinitiation complexes containing short nascent RNAs formed on the CTCCTC mutant were more sensitive to the effects of heparin and produced more abortive transcripts than similar complexes formed on SUP4-o. Our results suggest that the purine-rich sequences at the 5' ends of the nascent transcripts of many genes act to stabilize the early ternary complex.

scholarly journals Interactions between RNA polymerase and the core recognition element are a determinant of transcription start site selection

2016 ◽  
Vol 113 (21) ◽  
pp. E2899-E2905 ◽  
Author(s):  
Irina O. Vvedenskaya ◽  
Hanif Vahedian-Movahed ◽  
Yuanchao Zhang ◽  
Deanne M. Taylor ◽  
Richard H. Ebright ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Transcription Start Site ◽  
Transcription Initiation ◽  
Specific Protein ◽  
Start Site ◽  
Transcription Start ◽  
Transcription Bubble ◽  
Recognition Element

During transcription initiation, RNA polymerase (RNAP) holoenzyme unwinds ∼13 bp of promoter DNA, forming an RNAP-promoter open complex (RPo) containing a single-stranded transcription bubble, and selects a template-strand nucleotide to serve as the transcription start site (TSS). In RPo, RNAP core enzyme makes sequence-specific protein–DNA interactions with the downstream part of the nontemplate strand of the transcription bubble (“core recognition element,” CRE). Here, we investigated whether sequence-specific RNAP–CRE interactions affect TSS selection. To do this, we used two next-generation sequencing-based approaches to compare the TSS profile of WT RNAP to that of an RNAP derivative defective in sequence-specific RNAP–CRE interactions. First, using massively systematic transcript end readout, MASTER, we assessed effects of RNAP–CRE interactions on TSS selection in vitro and in vivo for a library of 47 (∼16,000) consensus promoters containing different TSS region sequences, and we observed that the TSS profile of the RNAP derivative defective in RNAP–CRE interactions differed from that of WT RNAP, in a manner that correlated with the presence of consensus CRE sequences in the TSS region. Second, using 5′ merodiploid native-elongating-transcript sequencing, 5′ mNET-seq, we assessed effects of RNAP–CRE interactions at natural promoters in Escherichia coli, and we identified 39 promoters at which RNAP–CRE interactions determine TSS selection. Our findings establish RNAP–CRE interactions are a functional determinant of TSS selection. We propose that RNAP–CRE interactions modulate the position of the downstream end of the transcription bubble in RPo, and thereby modulate TSS selection, which involves transcription bubble expansion or transcription bubble contraction (scrunching or antiscrunching).

A transcriptionally active tRNA gene interferes with nucleosome positioning in vivo

1992 ◽  
Vol 12 (9) ◽  
pp. 4015-4025
Author(s):  
R H Morse ◽  
S Y Roth ◽  
R T Simpson
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Trna Gene ◽  
Rna Polymerase Iii ◽  
Nucleosome Positioning ◽  
Yeast Cells ◽  
Trna Genes ◽  
Start Site ◽  
Cis Acting

Incorporation into a positioned nucleosome of a cis-acting element essential for replication in Saccharomyces cerevisiae disrupts the function of the element in vivo [R. T. Simpson, Nature (London) 343:387-389, 1990]. Furthermore, nucleosome positioning has been implicated in repression of transcription by RNA polymerase II in yeast cells. We have now asked whether the function of cis-acting elements essential for transcription of a gene transcribed by RNA polymerase III can be similarly affected. A tRNA gene was fused to either of two nucleosome positioning signals such that the predicted nucleosome would incorporate near its center the tRNA start site and essential A-box element. These constructs were then introduced into yeast cells on stably maintained, multicopy plasmids. Competent tRNA genes were transcribed in vivo and were not incorporated into positioned nucleosomes. Mutated, inactive tRNA genes were incorporated into nucleosomes whose positions were as predicted. This finding demonstrates that the transcriptional competence of the tRNA gene determined its ability to override a nucleosome positioning signal in vivo and establishes that a hierarchy exists between cis-acting elements and nucleosome positioning signals.

scholarly journals Termination-altering mutations in the second-largest subunit of yeast RNA polymerase III.

1995 ◽  
Vol 15 (3) ◽  
pp. 1467-1478 ◽  
Author(s):  
S A Shaaban ◽  
B M Krupp ◽  
B D Hall
Keyword(s):  
Amino Acid ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Transcription Termination ◽  
Rna Polymerase Iii ◽  
In Vitro Transcription ◽  
The Other ◽  
Polymerase Iii

In order to identify catalytically important amino acid changes within the second-largest subunit of yeast RNA polymerase III, we mutagenized selected regions of its gene (RET1) and devised in vivo assays for both increased and decreased transcription termination by this enzyme. Using as the reporter gene a mutant SUP4-o tRNA gene that in one case terminates prematurely and in the other case fails to terminate, we screened mutagenized RET1 libraries for reduced and increased transcription termination, respectively. The gain in suppression phenotype was in both cases scored as a reduction in the accumulation of red pigment in yeast strains harboring the ade2-1 ochre mutation. Termination-altering mutations were obtained in regions of the RET1 gene encoding amino acids 300 to 325, 455 to 486, 487 to 521, and 1061 to 1082 of the protein. In degree of amino acid sequence conservation, these range from highly variable in the first to highly conserved in the last two regions. Residues 300 to 325 yielded mainly reduced-termination mutants, while in region 1061 to 1082, increased-termination mutants were obtained exclusively. All mutants recovered, while causing gain of suppression with one SUP4 allele, brought about a reduction in suppression with the other allele, thus confirming that the phenotype is due to altered termination rather than an elevated level of transcription initiation. In vitro transcription reactions performed with extracts from several strong mutants demonstrated that the mutant polymerases respond to RNA terminator sequences in a manner that matches their in vivo termination phenotypes.

scholarly journals CK2 Forms a Stable Complex with TFIIIB and Activates RNA Polymerase III Transcription in Human Cells

2002 ◽  
Vol 22 (11) ◽  
pp. 3757-3768 ◽  
Author(s):  
Imogen M. Johnston ◽  
Simon J. Allison ◽  
Jennifer P. Morton ◽  
Laura Schramm ◽  
Pamela H. Scott ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Mammalian Cells ◽  
Rna Polymerase Iii ◽  
Stable Complex ◽  
Class Iii ◽  
Potent Effect ◽  
Cell Extracts ◽  
Polymerase Iii ◽  
Chemical Inhibitors

ABSTRACT CK2 is a highly conserved protein kinase with growth-promoting and oncogenic properties. It is known to activate RNA polymerase III (PolIII) transcription in Saccharomyces cerevisiae and is shown here to also exert a potent effect on PolIII in mammalian cells. Peptide and chemical inhibitors of CK2 block PolIII transcription in human cell extracts. Furthermore, PolIII transcription in mammalian fibroblasts is decreased significantly when CK2 activity is compromised by chemical inhibitors, antisense oligonucleotides, or kinase-inactive mutants. Coimmunoprecipitation and cofractionation show that endogenous human CK2 associates stably and specifically with the TATA-binding protein-containing factor TFIIIB, which brings PolIII to the initiation site of all class III genes. Serum stimulates TFIIIB phosphorylation in vivo, an effect that is diminished by inhibitors of CK2. Binding to TFIIIC2 recruits TFIIIB to most PolIII promoters; this interaction is compromised specifically by CK2 inhibitors. The data suggest that CK2 stimulates PolIII transcription by binding and phosphorylating TFIIIB and facilitating its recruitment by TFIIIC2. CK2 also activates PolI transcription in mammals and may therefore provide a mechanism to coregulate the output of PolI and PolIII. CK2 provides a rare example of an endogenous activity that operates on the PolIII system in both mammals and yeasts. Such evolutionary conservation suggests that this control may be of fundamental importance.

scholarly journals Mechanism of RNA polymerase III termination-associated reinitiation-recycling conferred by the essential function of the N terminal-and-linker domain of the C11 subunit

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Saurabh Mishra ◽  
Shaina H. Hasan ◽  
Rima M. Sakhawala ◽  
Shereen Chaudhry ◽  
Richard J. Maraia
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Point Mutations ◽  
Cleavage Activity ◽  
Terminal Domain ◽  
Polymerase Iii ◽  
Essential Function ◽  
High Level ◽  
Pol Iii

AbstractRNA polymerase III achieves high level tRNA synthesis by termination-associated reinitiation-recycling that involves the essential C11 subunit and heterodimeric C37/53. The C11-CTD (C-terminal domain) promotes Pol III active center-intrinsic RNA 3′-cleavage although deciphering function for this activity has been complicated. We show that the isolated NTD (N-terminal domain) of C11 stimulates Pol III termination by C37/53 but not reinitiation-recycling which requires the NTD-linker (NTD-L). By an approach different from what led to current belief that RNA 3′-cleavage activity is essential, we show that NTD-L can provide the essential function of Saccharomyces cerevisiae C11 whereas classic point mutations that block cleavage, interfere with active site function and are toxic to growth. Biochemical and in vivo analysis including of the C11 invariant central linker led to a model for Pol III termination-associated reinitiation-recycling. The C11 NTD and CTD stimulate termination and RNA 3′-cleavage, respectively, whereas reinitiation-recycling activity unique to Pol III requires only the NTD-linker. RNA 3′-cleavage activity increases growth rate but is nonessential.

Sign in / Sign up

Export Citation Format

Share Document