scholarly journals Additional intragenic promoter elements of the Xenopus 5S RNA genes upstream from the TFIIIA-binding site.

1990 ◽  
Vol 10 (10) ◽  
pp. 5166-5176 ◽  
Author(s):  
H J Keller ◽  
Q M You ◽  
P J Romaniuk ◽  
J M Gottesfeld
Keyword(s):  
Binding Site ◽  
Transcription Initiation ◽  
Rna Polymerase Iii ◽  
Initiation Factor ◽  
Coding Region ◽  
5S Rna ◽  
Promoter Elements ◽  
Substitution Mutations ◽  
Type Gene ◽  
5S Gene

The major promoter element of the Xenopus laevis 5S RNA gene is located within the transcribed region of the gene and forms the binding site for the transcription initiation factor TFIIIA. We report an analysis of deletion and substitution mutations within the coding region of the major oocyte-type 5S gene of X. laevis. Our results differ from those of previous mutagenesis studies conducted on the somatic-type genes of Xenopus borealis and X. laevis. Transcription assays in whole oocyte S-150 extracts, with both oocyte- and somatic-type mutants, revealed additional promoter elements between the start site for transcription and the binding site for TFIIIA. These sequences regulate the efficiency of binding TFIIIC, a transcription factor required by the genes transcribed by RNA polymerase III containing intragenic promoters. Under TFIIIC-limiting conditions, the somatic-type gene had a 10-fold-higher affinity for TFIIIC than did the major oocyte-type 5S gene. One mutation in the oocyte-type gene (nucleotides +33 to +39) reduced TFIIIC affinity and transcriptional activity four- to fivefold. Differences in TFIIIC affinity between oocyte- and somatic-type genes may contribute to the differential transcription of these genes observed during Xenopus embryogenesis.

Additional intragenic promoter elements of the Xenopus 5S RNA genes upstream from the TFIIIA-binding site

1990 ◽  
Vol 10 (10) ◽  
pp. 5166-5176
Author(s):  
H J Keller ◽  
Q M You ◽  
P J Romaniuk ◽  
J M Gottesfeld
Keyword(s):  
Binding Site ◽  
Transcription Initiation ◽  
Rna Polymerase Iii ◽  
Initiation Factor ◽  
Coding Region ◽  
5S Rna ◽  
Promoter Elements ◽  
Substitution Mutations ◽  
Type Gene ◽  
5S Gene

The major promoter element of the Xenopus laevis 5S RNA gene is located within the transcribed region of the gene and forms the binding site for the transcription initiation factor TFIIIA. We report an analysis of deletion and substitution mutations within the coding region of the major oocyte-type 5S gene of X. laevis. Our results differ from those of previous mutagenesis studies conducted on the somatic-type genes of Xenopus borealis and X. laevis. Transcription assays in whole oocyte S-150 extracts, with both oocyte- and somatic-type mutants, revealed additional promoter elements between the start site for transcription and the binding site for TFIIIA. These sequences regulate the efficiency of binding TFIIIC, a transcription factor required by the genes transcribed by RNA polymerase III containing intragenic promoters. Under TFIIIC-limiting conditions, the somatic-type gene had a 10-fold-higher affinity for TFIIIC than did the major oocyte-type 5S gene. One mutation in the oocyte-type gene (nucleotides +33 to +39) reduced TFIIIC affinity and transcriptional activity four- to fivefold. Differences in TFIIIC affinity between oocyte- and somatic-type genes may contribute to the differential transcription of these genes observed during Xenopus embryogenesis.

scholarly journals Inhibition of TATA Binding Protein Dimerization by RNA Polymerase III Transcription Initiation Factor Brf1

2004 ◽  
Vol 279 (31) ◽  
pp. 32401-32406 ◽  
Author(s):  
Diane E. Alexander ◽  
David J. Kaczorowski ◽  
Amy J. Jackson-Fisher ◽  
Drew M. Lowery ◽  
Sara J. Zanton ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
Binding Protein ◽  
Rna Polymerase Iii ◽  
Tata Binding Protein ◽  
Initiation Factor ◽  
Protein Dimerization ◽  
Polymerase Iii ◽  
Transcription Initiation Factor

scholarly journals Structural basis of transcription inhibition by fidaxomicin (lipiarmycin A3)

2017 ◽  
Author(s):  
Wei Lin ◽  
Kalyan Das ◽  
David Degen ◽  
Abhishek Mazumder ◽  
Diego Duchi ◽  
...  
Keyword(s):  
Binding Site ◽  
Single Molecule ◽  
Transcription Initiation ◽  
Resonance Energy ◽  
Active Pharmaceutical Ingredient ◽  
Conformational State ◽  
Initiation Factor ◽  
Structural Basis ◽  
Cross Resistance ◽  
Antibacterial Drug

Fidaxomicin is an antibacterial drug in clinical use in treatment ofClostridium difficilediarrhea1–2. The active pharmaceutical ingredient of fidaxomicin, lipiarmycin A3 (Lpm)1–4, is a macrocyclic antibiotic with bactericidal activity against Gram-positive bacteria and efflux-deficient strains of Gram-negative bacteria1–2, 5. Lpm functions by inhibiting bacterial RNA polymerase (RNAP)6–8. Lpm exhibits no cross-resistance with the classic RNAP inhibitor rifampin (Rif)7, 9and inhibits transcription initiation at an earlier step than Rif8–11, suggesting that the binding site and mechanism of Lpm differ from those of Rif. Efforts spanning a decade to obtain a crystal structure of RNAP in complex with Lpm have been unsuccessful. Here, we report a cryo-EM12–13structure ofMycobacterium tuberculosisRNAP holoenzyme in complex with Lpm at 3.5 Å resolution. The structure shows that Lpm binds at the base of the RNAP “clamp,” interacting with the RNAP switch region and the RNAP RNA exit channel. The binding site on RNAP for Lpm does not overlap the binding sites for other RNAP inhibitors, accounting for the absence of cross-resistance of Lpm with other RNAP inhibitors. The structure exhibits an open conformation of the RNAP clamp, with the RNAP clamp swung outward by ~17° relative to its position in catalytically competent RNAP-promoter transcription initiation complexes, suggesting that Lpm traps an open-clamp conformational state. Single-molecule fluorescence resonance energy transfer14experiments confirm that Lpm traps an open-clamp conformational state and define effects of Lpm on clamp opening and closing dynamics. We propose that Lpm inhibits transcription initiation by trapping an open-clamp conformational state, thereby preventing simultaneous engagement of transcription initiation factor σ regions 2 and 4 with promoter -10 and -35 elements. The results provide information essential to understanding the mode of action of Lpm, account for structure-activity relationships of known Lpm analogs, and suggest modifications to Lpm that could yield new, improved Lpm analogs.

scholarly journals Structural basis of RNA polymerase III transcription repression by Maf1

2019 ◽  
Author(s):  
Matthias K. Vorländer ◽  
Florence Baudin ◽  
Robyn D. Moir ◽  
René Wetzel ◽  
Wim J. H. Hagen ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Binding Site ◽  
Transcription Initiation ◽  
Rna Polymerase Iii ◽  
Structural Basis ◽  
Metabolic Efficiency ◽  
Transcription Repression ◽  
Polymerase Iii ◽  
Wide Range ◽  
Pol Iii

ABSTRACTMaf1 is a highly conserved central regulator of transcription by RNA polymerase III (Pol III), and Maf1 activity influences a wide range of phenotypes from metabolic efficiency to lifespan. Here, we present a 3.3 Å cryo-EM structure of yeast Maf1 bound to Pol III, which establishes how Maf1 achieves transcription repression. In the Maf1-bound state, Pol III elements that are involved in transcription initiation are sequestered, and the active site is sealed off due to ordering of the mobile C34 winged helix 2 domain. Specifically, the Maf1 binding site overlaps with the binding site of the Pol III transcription factor TFIIIB and DNA in the pre-initiation complex, rationalizing that binding of Maf1 and TFIIIB to Pol III are mutually exclusive. We validate our structure using variants of Maf1 with impaired transcription-inhibition activity. These results reveal the exact mechanism of Pol III inhibition by Maf1, and rationalize previous biochemical data.

scholarly journals Formation of an active transcription complex in the Drosophila melanogaster 5S RNA gene is dependent on an upstream region.

1987 ◽  
Vol 7 (6) ◽  
pp. 2046-2051 ◽  
Author(s):  
A D Garcia ◽  
A M O'Connell ◽  
S J Sharp
Keyword(s):  
Drosophila Melanogaster ◽  
Transcription Initiation ◽  
Rna Polymerase Iii ◽  
In Vitro Transcription ◽  
Upstream Region ◽  
Nucleotide Position ◽  
Wild Type ◽  
5S Rna ◽  
Cell Extracts

We constructed deletion-substitution and linker-scanning mutations in the 5'-flanking region of the Drosophila melanogaster 5S RNA gene. In vitro transcription of these templates in Drosophila and HeLa cell extracts revealed the presence of an essential control region (-30 region) located between nucleotides -39 and -26 upstream of the transcription initiation site: deletion of sequences upstream of nucleotide position -39 had no detectable effect on the wild-type level of in vitro transcription, whereas mutations extending between positions -39 and 1 resulted in templates with decreased transcriptional levels; specifically, deletion and linker-scanning mutations in the -34 to -26 region (-30 region) resulted in loss of transcription. The -30 region is essential for transcription and therefore forms part of the Drosophila 5S RNA gene transcription promoter. Compared with the activity of the wild-type gene, mutant 5S DNAs exhibited no impairment in the ability to sequester limiting transcription factors in a template exclusion competition assay. While we do not know which transcription factor(s) interacts with the -30 region, the possible involvement of RNA polymerase III at this region is discussed.

scholarly journals Cloning of the human erythropoietin receptor gene

Blood ◽  
1991 ◽  
Vol 78 (10) ◽  
pp. 2548-2556 ◽  
Author(s):  
CT Noguchi ◽  
KS Bae ◽  
K Chin ◽  
Y Wada ◽  
AN Schechter ◽  
...  
Keyword(s):  
Binding Site ◽  
Translational Control ◽  
Transcription Initiation ◽  
Repetitive Sequences ◽  
Cdna Probe ◽  
Gc Content ◽  
Erythropoietin Receptor ◽  
Coding Region ◽  
Epo Receptor ◽  
Human Erythropoietin

We have isolated and characterized a genomic clone of the human erythropoietin (Epo) receptor from a placental genomic library using a cDNA probe for the murine Epo receptor. The coding region spans about 6.5 kb with seven intervening sequences ranging in size from 81 bp to 2.1 kb. A stretch of 123 purines is found in the 5′ region from -456 to -578 upstream from the first codon and flanking the Alu repetitive sequences located further upstream. The human Epo receptor contains a palindromic sequence 5′ of the translated region that consists of an almost perfect inverted repeat of 12 nucleotides (CAGCTGC(G/C)TCCG) centered about G at -92 from the first codon. An inverted SP1 binding site (CCGCCC) and an inverted GATA-1 binding site (TTATCT) are located at positions -151 and -179, respectively, and CACCC sequences are located at -585 and further upstream. No TATA or CAAT sequences are in this 5′ flanking region. However, this region as far as -275 has a 72% GC content compared with an overall GC content of 56%. A 1-kb BamHI fragment of the human Epo receptor containing 700 bp of sequences 5′ of the coding region was transcribed in an in vitro transcription assay; initiation of transcription appeared to be around 132 +/- 5 just downstream from the inverted SP1 site at -151. T1 analysis of human Epo receptor messenger RNA also maps the site of transcription initiation to this region. Within 180 nucleotides 5′ to the first exon are three regions with 70% or greater homology with the murine Epo receptor. The study of this gene, including its similarities with the murine Epo receptor, should help elucidate aspects of the transcriptional and possible translational control of the Epo receptor in human erythroid cells and thus its role in signal transduction and erythroid differentiation.

scholarly journals RNA Polymerase III Subunit Mutations in Genetic Diseases

2021 ◽  
Vol 8 ◽  
Author(s):  
Elisabeth Lata ◽  
Karine Choquet ◽  
Francis Sagliocco ◽  
Bernard Brais ◽  
Geneviève Bernard ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Current Knowledge ◽  
Rna Polymerase Iii ◽  
Initiation Factor ◽  
Varicella Zoster ◽  
Vital Functions ◽  
Genes Encoding ◽  
Pol Iii

RNA polymerase (Pol) III transcribes small untranslated RNAs such as 5S ribosomal RNA, transfer RNAs, and U6 small nuclear RNA. Because of the functions of these RNAs, Pol III transcription is best known for its essential contribution to RNA maturation and translation. Surprisingly, it was discovered in the last decade that various inherited mutations in genes encoding nine distinct subunits of Pol III cause tissue-specific diseases rather than a general failure of all vital functions. Mutations in the POLR3A, POLR3C, POLR3E and POLR3F subunits are associated with susceptibility to varicella zoster virus-induced encephalitis and pneumonitis. In addition, an ever-increasing number of distinct mutations in the POLR3A, POLR3B, POLR1C and POLR3K subunits cause a spectrum of neurodegenerative diseases, which includes most notably hypomyelinating leukodystrophy. Furthermore, other rare diseases are also associated with mutations in genes encoding subunits of Pol III (POLR3H, POLR3GL) and the BRF1 component of the TFIIIB transcription initiation factor. Although the causal relationship between these mutations and disease development is widely accepted, the exact molecular mechanisms underlying disease pathogenesis remain enigmatic. Here, we review the current knowledge on the functional impact of specific mutations, possible Pol III-related disease-causing mechanisms, and animal models that may help to better understand the links between Pol III mutations and disease.

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