Activation of RNA polymerase III transcription of human Alu repetitive elements by adenovirus type 5: requirement for the E1b 58-kilodalton protein and the products of E4 open reading frames 3 and 6

1993 ◽  
Vol 13 (6) ◽  
pp. 3231-3244
Author(s):  
B Panning ◽  
J R Smiley
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Adenovirus Type ◽  
Open Reading Frames ◽  
Alu Elements ◽  
Polymerase Iii ◽  
Novel Approach ◽  
Adenovirus E1b ◽  
Adenovirus Type 5 ◽  
Reading Frames

We found that transcription of endogenous human Alu elements by RNA polymerase III was strongly stimulated following infection of HeLa cells with adenovirus type 5, leading to the accumulation of high levels of Alu transcripts initiated from Alu polymerase III promoters. In contrast to previously reported cases of adenovirus-induced activation of polymerase III transcription, induction required the E1b 58-kDa protein and the products of E4 open reading frames 3 and 6 in addition to the 289-residue E1a protein. In addition, E1a function was not required at high multiplicities of infection, suggesting that E1a plays an indirect role in Alu activation. These results suggest previously unsuspected regulatory properties of the adenovirus E1b and E4 gene products and provide a novel approach to the study of the biology of the most abundant class of dispersed repetitive DNA in the human genome.

scholarly journals Activation of RNA polymerase III transcription of human Alu repetitive elements by adenovirus type 5: requirement for the E1b 58-kilodalton protein and the products of E4 open reading frames 3 and 6.

1993 ◽  
Vol 13 (6) ◽  
pp. 3231-3244 ◽  
Author(s):  
B Panning ◽  
J R Smiley
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Adenovirus Type ◽  
Open Reading Frames ◽  
Alu Elements ◽  
Polymerase Iii ◽  
Novel Approach ◽  
Adenovirus E1b ◽  
Adenovirus Type 5 ◽  
Reading Frames

We found that transcription of endogenous human Alu elements by RNA polymerase III was strongly stimulated following infection of HeLa cells with adenovirus type 5, leading to the accumulation of high levels of Alu transcripts initiated from Alu polymerase III promoters. In contrast to previously reported cases of adenovirus-induced activation of polymerase III transcription, induction required the E1b 58-kDa protein and the products of E4 open reading frames 3 and 6 in addition to the 289-residue E1a protein. In addition, E1a function was not required at high multiplicities of infection, suggesting that E1a plays an indirect role in Alu activation. These results suggest previously unsuspected regulatory properties of the adenovirus E1b and E4 gene products and provide a novel approach to the study of the biology of the most abundant class of dispersed repetitive DNA in the human genome.

scholarly journals Unusual Properties of Adenovirus E2E Transcription by RNA Polymerase III

2003 ◽  
Vol 77 (7) ◽  
pp. 4015-4024 ◽  
Author(s):  
Wenlin Huang ◽  
S. J. Flint
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Polymerase Iii ◽  
Adenovirus Type ◽  
Transcription Unit ◽  
Promoter Sequences ◽  
Polymerase Iii ◽  
Infected Cells ◽  
Adenovirus Type 5 ◽  
Rna Polymerase Ii Transcription

ABSTRACT In adenovirus type 5-infected cells, RNA polymerase III transcription of a gene superimposed on the 5′ end of the E2E RNA polymerase II transcription unit produces two small (<100-nucleotide) RNAs that accumulate to low steady-state concentrations (W. Huang, R. Pruzan, and S. J. Flint, Proc. Natl. Acad. Sci. USA 91:1265-1269, 1984). To gain a better understanding of the function of this RNA polymerase III transcription, we have examined the properties of the small E2E RNAs and E2E RNA polymerase III transcription in more detail. The accumulation of cytoplasmic E2E RNAs and the rates of E2E transcription by the two RNA polymerases during the infectious cycle were analyzed by using RNase T1 protection and run-on transcription assays, respectively. Although the RNA polymerase III transcripts were present at significantly lower concentrations than E2E mRNA throughout the period examined, E2E transcription by RNA polymerase III was found to be at least as efficient as that by RNA polymerase II. The short half-lifes of the small E2E RNAs estimated by using the actinomycin D chase method appear to account for their limited accumulation. The transcription of E2E sequences by RNA polymerase II and III in cells infected by recombinant adenoviruses carrying ectopic E2E-CAT (chloramphenicol transferase) reporter genes with mutations in E2E promoter sequences was also examined. The results of these experiments indicate that recognition of the E2E promoter by the RNA polymerase II transcriptional machinery in infected cells limits transcription by RNA polymerase III, and vice versa. Such transcriptional competition and the properties of E2E RNAs made by RNA polymerase III suggest that the function of this viral RNA polymerase III transcription unit is unusual.

The RNA polymerase III terminator used by a B1-Alu element can modulate 3' processing of the intermediate RNA product

1992 ◽  
Vol 12 (4) ◽  
pp. 1500-1506
Author(s):  
R J Maraia ◽  
D Y Chang ◽  
A P Wolffe ◽  
R L Vorce ◽  
K Hsu
Keyword(s):  
Rna Polymerase ◽  
Dna Sequences ◽  
Rna Processing ◽  
Rna Polymerase Iii ◽  
Protein S ◽  
Nuclear Extract ◽  
Mouse Tissue ◽  
Alu Elements ◽  
Polymerase Iii ◽  
Nuclear Extracts

The dispersion of short interspersed elements (SINEs) probably occurred through an RNA intermediate. B1 is a murine homolog of the human SINE Alu; these elements are composed of 5' G + C-rich regions juxtaposed to A-rich tracts and are flanked by direct repeats. Internal promoters direct RNA polymerase III to transcribe B1 and Alu elements and proceed into the 3' flanking DNA until it reaches a (dT)4 termination signal. The resulting transcripts contain 3'-terminal oligo(U) tracts which can presumably base pair with the A-rich tract to form self-primed templates for reverse transcriptase and retrotransposition. Nuclear extracts from mouse tissue culture cells contain an RNA processing activity that removes the A-rich and 3'-terminal regions from purified B1 RNAs (R. Maraia, Nucleic Acids Res. 19:5695-5702, 1991). In this study, we examined transcription and RNA processing in these nuclear extracts. In contrast to results with use of purified RNA, nascent transcripts synthesized in nuclear extract by RNA polymerase III are not processed, suggesting that the transposition-intermediate-like RNA is shielded from processing by a protein(s). Alteration of an AATTTT TAA termination signal to a GCTTTTGC signal activated processing by greater than 100-fold in coupled transcription/processing reactions. A similar difference was found when expression was compared in frog oocytes. No difference in processing was found if the transcripts were made by T7 RNA polymerase in the presence of the nuclear extract, indicating that the different processing effects of the two terminators were dependent on synthesis by polymerase III. The modulation of processing of B1-Alu transcripts and the potential for retrotransposition of B1 and Alu DNA sequences are discussed.

scholarly journals Rapid enrichment of HeLa transcription factors IIIB and IIIC by using affinity chromatography based on avidin-biotin interactions.

1986 ◽  
Vol 6 (9) ◽  
pp. 3117-3127 ◽  
Author(s):  
M S Kasher ◽  
D Pintel ◽  
D C Ward
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase ◽  
Specific Activity ◽  
Rna Polymerase Iii ◽  
Adenovirus Type ◽  
Stable Complex ◽  
Polymerase Iii ◽  
S100 Extract ◽  
Affinity Resin ◽  
A Site

Plasmid DNA containing the adenovirus type 2 genes for VA RNA was linearized at a site distal to the gene, end labeled with a biotin-nucleotide analog of TTP, and incubated with avidin to form an avidin-biotinylated DNA complex. HeLa cell S100 extracts containing crude RNA polymerase III and transcription factors (TFs) IIIB and IIIC were programmed with the avidin-biotin-VA DNA to allow stable complex formation (A.B. Lassar, P.L. Martin, and R.G. Roeder, Science 222:740-748, 1983). Chromatography of the programmed extract over a biotin-cellulose affinity resin resulted in the selective, and virtually quantitative, retention of one of two stable preinitiation complexes, either VA-IIIC or VA-IIIC-IIIB, depending on the length of template incubation in the S100 extract. After washing the resin with 0.10 M and 0.25 M KCl to remove RNA polymerase III and nonspecifically bound proteins, respectively, TFIIIC was eluted from the VA-IIIC complex by the addition of 1.5 M KCl. The VA-IIIC-IIIB complex exhibited a higher salt stability. Most of TFIIIB and some TFIIIC were released by the addition of 1.5 M KCl; however, the majority of TFIIIC activity was recovered only after a subsequent 3.0 M KCl elution. The specific activity of the TFIIIC in the 3.0 M KCl fraction was 770-fold higher than that in the S100 extract, while the protein content of the 1.5 and 3.0 M KCl fractions was reduced 7,500- and 100,000-fold, respectively.

scholarly journals Dimethylation of Histone H3 at Lysine 36 DemarcatesRegulatory and Nonregulatory ChromatinGenome-Wide

2005 ◽  
Vol 25 (21) ◽  
pp. 9447-9459 ◽  
Author(s):  
Bhargavi Rao ◽  
Yoichiro Shibata ◽  
Brian D. Strahl ◽  
Jason D. Lieb
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Nucleosome Occupancy ◽  
Rna Polymerase Iii ◽  
Histone H3 ◽  
Open Reading Frames ◽  
Gene Transcript ◽  
Genome Wide ◽  
Rnap Ii ◽  
Reading Frames

ABSTRACT Set2p, which mediates histone H3 lysine 36 dimethylation (H3K36me2) in Saccharomyces cerevisiae, has been shown to associate with RNA polymerase II (RNAP II) at individual loci. Here, chromatin immunoprecipitation-microarray experiments normalized to general nucleosome occupancy reveal that nucleosomes within open reading frames (ORFs) and downstream noncoding chromatin were highly dimethylated at H3K36 and that Set2p activity begins at a stereotypic distance from the initiation of transcription genome-wide. H3K36me2 is scarce in regions upstream of divergently transcribed genes, telomeres, silenced mating loci, and regions transcribed by RNA polymerase III, providing evidence that the enzymatic activity of Set2p is restricted to its association with RNAP II. The presence of H3K36me2 within ORFs correlated with the “on” or“ off” state of transcription, but the degree of H3K36 dimethylation within ORFs did not correlate with transcription frequency. This provides evidence that H3K36me2 is established during the initial instances of gene transcription, with subsequent transcription having at most a maintenance role. Accordingly, newly activated genes acquire H3K36me2 in a manner that does not correlate with gene transcript levels. Finally, nucleosomes dimethylated at H3K36 appear to be refractory to loss from highly transcribed chromatin. Thus, H3K36me2, which is highly conserved throughout eukaryotic evolution, provides a stable molecular mechanism for establishing chromatin context throughout the genome by distinguishing potential regulatory regions from transcribed chromatin.

scholarly journals The RNA polymerase III terminator used by a B1-Alu element can modulate 3' processing of the intermediate RNA product.

1992 ◽  
Vol 12 (4) ◽  
pp. 1500-1506 ◽  
Author(s):  
R J Maraia ◽  
D Y Chang ◽  
A P Wolffe ◽  
R L Vorce ◽  
K Hsu
Keyword(s):  
Rna Polymerase ◽  
Dna Sequences ◽  
Rna Processing ◽  
Rna Polymerase Iii ◽  
Protein S ◽  
Nuclear Extract ◽  
Mouse Tissue ◽  
Alu Elements ◽  
Polymerase Iii ◽  
Nuclear Extracts

The dispersion of short interspersed elements (SINEs) probably occurred through an RNA intermediate. B1 is a murine homolog of the human SINE Alu; these elements are composed of 5' G + C-rich regions juxtaposed to A-rich tracts and are flanked by direct repeats. Internal promoters direct RNA polymerase III to transcribe B1 and Alu elements and proceed into the 3' flanking DNA until it reaches a (dT)4 termination signal. The resulting transcripts contain 3'-terminal oligo(U) tracts which can presumably base pair with the A-rich tract to form self-primed templates for reverse transcriptase and retrotransposition. Nuclear extracts from mouse tissue culture cells contain an RNA processing activity that removes the A-rich and 3'-terminal regions from purified B1 RNAs (R. Maraia, Nucleic Acids Res. 19:5695-5702, 1991). In this study, we examined transcription and RNA processing in these nuclear extracts. In contrast to results with use of purified RNA, nascent transcripts synthesized in nuclear extract by RNA polymerase III are not processed, suggesting that the transposition-intermediate-like RNA is shielded from processing by a protein(s). Alteration of an AATTTT TAA termination signal to a GCTTTTGC signal activated processing by greater than 100-fold in coupled transcription/processing reactions. A similar difference was found when expression was compared in frog oocytes. No difference in processing was found if the transcripts were made by T7 RNA polymerase in the presence of the nuclear extract, indicating that the different processing effects of the two terminators were dependent on synthesis by polymerase III. The modulation of processing of B1-Alu transcripts and the potential for retrotransposition of B1 and Alu DNA sequences are discussed.

Rapid enrichment of HeLa transcription factors IIIB and IIIC by using affinity chromatography based on avidin-biotin interactions

1986 ◽  
Vol 6 (9) ◽  
pp. 3117-3127
Author(s):  
M S Kasher ◽  
D Pintel ◽  
D C Ward
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase ◽  
Specific Activity ◽  
Rna Polymerase Iii ◽  
Adenovirus Type ◽  
Stable Complex ◽  
Polymerase Iii ◽  
S100 Extract ◽  
Affinity Resin ◽  
A Site

Plasmid DNA containing the adenovirus type 2 genes for VA RNA was linearized at a site distal to the gene, end labeled with a biotin-nucleotide analog of TTP, and incubated with avidin to form an avidin-biotinylated DNA complex. HeLa cell S100 extracts containing crude RNA polymerase III and transcription factors (TFs) IIIB and IIIC were programmed with the avidin-biotin-VA DNA to allow stable complex formation (A.B. Lassar, P.L. Martin, and R.G. Roeder, Science 222:740-748, 1983). Chromatography of the programmed extract over a biotin-cellulose affinity resin resulted in the selective, and virtually quantitative, retention of one of two stable preinitiation complexes, either VA-IIIC or VA-IIIC-IIIB, depending on the length of template incubation in the S100 extract. After washing the resin with 0.10 M and 0.25 M KCl to remove RNA polymerase III and nonspecifically bound proteins, respectively, TFIIIC was eluted from the VA-IIIC complex by the addition of 1.5 M KCl. The VA-IIIC-IIIB complex exhibited a higher salt stability. Most of TFIIIB and some TFIIIC were released by the addition of 1.5 M KCl; however, the majority of TFIIIC activity was recovered only after a subsequent 3.0 M KCl elution. The specific activity of the TFIIIC in the 3.0 M KCl fraction was 770-fold higher than that in the S100 extract, while the protein content of the 1.5 and 3.0 M KCl fractions was reduced 7,500- and 100,000-fold, respectively.

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