Correlation between the activity of a 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole-insensitive puff and the synthesis of major heat-shock polypeptide, hsp70, in Chironomus thummi

1988 ◽  
Vol 66 (11) ◽  
pp. 1177-1185 ◽  
Author(s):  
D. Barettino ◽  
G. Morcillo ◽  
J. L. Díez ◽  
M. T. Carretero ◽  
M. J. Carmona
Keyword(s):  
Heat Shock ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Specific Inhibitor ◽  
In Vitro Transcription ◽  
The Other ◽  
Low Salt ◽  
Transcription Inhibitor ◽  
Chironomus Thummi

The induction of puff III-A3b, a major heat-shock puff in Chironomus thummi salivary cells, was insensitive to the transcription inhibitor 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB), whereas no transcriptional activity could be detected at the other heat-shock puffs in the presence of this drug. In these conditions, a polypeptide with the same Mr and isoform pattern as those of the major heat-shock polypeptide, hsp70, was synthesized. These results suggest that hsp70 is encoded by locus III-A3b. In addition to DRB insensitivity, incorporation of [3H]UTP on puff III-A3b took place in an in vitro transcription assay under low-salt conditions (100 mM NaCl); no labelling could be detected at the other heat-shock puffs under these conditions. Although DRB has been reported as a specific inhibitor of RNA polymerase II-directed transcription, and although the low-salt conditions were not propitious for the activity of this enzyme, RNA polymerase II was detected on puff III-A3b and on the other heat-shock puffs by immunofluorescence with anti-RNA polymerase II antibodies.

scholarly journals Bacteriophage SP6-specific RNA polymerase. II. Mapping of SP6 DNA and selective in vitro transcription.

1982 ◽  
Vol 257 (10) ◽  
pp. 5779-5788 ◽  
Author(s):  
G A Kassavetis ◽  
E T Butler ◽  
D Roulland ◽  
M J Chamberlin
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
In Vitro Transcription

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 A novel, non-radioactive eukaryotic in vitro transcription assay for sensitive quantification of RNA polymerase II activity

2014 ◽  
Vol 15 (1) ◽  
pp. 7 ◽  
Author(s):  
Cristina Voss ◽  
Brita Schmitt ◽  
Susanne Werner-Simon ◽  
Christian Lutz ◽  
Werner Simon ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
In Vitro Transcription ◽  
Transcription Assay

scholarly journals A mouse in vitro transcription system reconstituted from highly purified RNA polymerase II, TFIIH and recombinant TBP, TFIIB, TFIIE and TFIIF

2001 ◽  
Vol 268 (16) ◽  
pp. 4527-4536 ◽  
Author(s):  
Irina Kotova ◽  
Anna Lena Chabes ◽  
Bo Segerman ◽  
Sara Flodell ◽  
Lars Thelander ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
In Vitro Transcription ◽  
Transcription System

scholarly journals Anatomy of an unusual RNA polymerase II promoter containing a downstream TATA element.

1992 ◽  
Vol 12 (6) ◽  
pp. 2884-2897 ◽  
Author(s):  
Y Kasai ◽  
H Chen ◽  
S J Flint
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Mutational Analysis ◽  
Adenovirus Type ◽  
In Vitro Transcription ◽  
Cell Extracts ◽  
Tata Element ◽  
Tata Sequence

The adenovirus type 2 IVa2 promoter lacks a conventional TATA element yet directs transcription from two closely spaced initiation sites. To define elements required for in vitro transcription of this promoter, IVa2 templates carrying 5' deletions or linker-scanning mutations were transcribed in HeLa whole-cell extracts and the transcripts were analyzed by primer extension. Mutation of the sequence centered on position -47, which is specifically recognized by a cellular factor, reduced the efficiency of IVa2 transcription two- to threefold, whereas mutation of the sequence centered on position -30 selectively impaired utilization of the minor in vivo initiation site. Utilization of the major in vivo site was decreased no more than fivefold by deletion of all sequences upstream of position -15. By contrast, mutation of the region from +13 to +19 or of the initiation region severely impaired IVa2 transcription. The sequence spanning the initiation sites was sufficient to direct accurate initiation by RNA polymerase II from the major in vivo site. Thus, the two initiation sites of the IVa2 promoter are specified by independent elements, and a downstream element is the primary determinant of efficient transcription from both of these sites. The downstream element identified by mutational analysis altered the TATA element-like sequence TATAGAAA lying at positions +21 to +14 in the coding strand. Transcription from the wild-type IVa2 promoter was severely inhibited when endogenous TFIID was inactivated by mild heat treatment. Exogenous human TATA-binding protein (TBP) synthesized in Escherichia coli restored specific IVa2 transcription from both initiation sites when added to such heat-treated extracts. Although efficient IVa2 transcription requires both the downstream TATA sequence and active TFIID, bacterially synthesized TBP also stimulated the low level of IVa2 transcription observed when the TATA sequence was mutated to a sequence that failed to bind TBP.

scholarly journals Polyphasic changes in incorporation of precursors into ribonucleic acid of oestradiol-stimulated mammary gland

1975 ◽  
Vol 152 (2) ◽  
pp. 211-216 ◽  
Author(s):  
Ferdinando Auricchio ◽  
Rocco Satriano ◽  
Andrea Rotondi ◽  
Ada Quirino ◽  
Francesco Bresciani
Keyword(s):  
Mammary Gland ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
High Salt ◽  
Precursor Pool ◽  
Ovariectomized Mice ◽  
Low Salt ◽  
Time Point

1. At 3 weeks after ovariectomy, mammary glands (5th pair) of adult Swiss mice show (i) no significant decrease in weight, (ii) 20% of the original rate of incorporation of [3H]-uridine into RNA (after a 30min pulse), and (iii) 90% of the original rate of incorporation of l-[3H]leucine into protein (after a 15min pulse). 2. A single injection of oestradiol-17β into these ovariectomized mice produces, during the next 17h, a series of discrete bursts of increased incorporation of [3H]uridine into mammary-gland RNA; the bursts, which are variable in height, reach peaks at approx. 1, 9, 12 and 16h after hormone administration; an increase is already detected at 15min, the earliest time-point investigated; each burst lasts for approx. 2h. There is no significant stimulation of [3H]uridine incorporation into RNA of liver and quadriceps femoris muscle. 3. Nuclear incorporation of [3H]UTP into RNA of mammary gland in vitro is linear with time for up to 20min at 15°C; it requires CTP, GTP and ATP and is inhibited by actinomycin D. Also, the incorporation is strongly inhibited by α-amanitin in high salt concentrations but only weakly in low salt concentrations, a result indicating that RNA polymerase II activity predominates in high salt, whereas RNA polymerase I activity predominates in low salt concentrations. Injection of oestradiol-17β in vivo followed by measurement of nuclear RNA synthesis in vitro shows a definite increase in both RNA polymerase activities 30min after oestradiol-17β injection, the earliest time-point investigated, a higher increase at 1h, a decline at 4h, and again a large increase at 12h. These results in general agree with the changes in precursor incorporation into RNA measured directly in the animal and suggest that changes in [3H]uridine uptake into RNA are not precursor-pool-dependent.

scholarly journals Structural and Functional Characterization of PC2 and RNA Polymerase II-Associated Subpopulations of Metazoan Mediator

2005 ◽  
Vol 25 (6) ◽  
pp. 2117-2129 ◽  
Author(s):  
Sohail Malik ◽  
Hwa Jin Baek ◽  
Weizhen Wu ◽  
Robert G. Roeder
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Specific Activity ◽  
Functional Characterization ◽  
In Vitro Transcription ◽  
Dynamic Nature ◽  
Pol Ii ◽  
General Transcription Factors

ABSTRACT The coactivator complexes TRAP/SMCC and PC2 represent two forms of Mediator. To further understand the implications of the heterogeneity of the cellular Mediator populations for regulation of RNA polymerase II (Pol II) transcription, we used a combination of affinity and conventional chromatographic methods. Our analysis revealed a spectrum of complexes, including some containing significant proportions of Pol II. Interestingly, the subunit composition of the Pol II-associated Mediator population resembled that of PC2 more closely than that of the larger TRAP/SMCC complex. In in vitro transcription assays reconstituted from homogeneous preparations of general transcription factors, Mediator-associated Pol II displayed a greater specific activity (relative to that of standard Pol II) in activator-independent (basal) transcription in addition to the previously described effects of Mediator on activator-dependent transcription. Purified PC2 complex also stimulated basal activity under these conditions. Immobilized template assays in which activator-recruited preinitiation complexes were allowed to undergo one cycle of transcription revealed partial disruption of Mediator that resulted in a PC2-like complex being retained in the scaffold. This result implies that PC2 could originate as a result of a normal cellular process. Our results are thus consistent with a dynamic nature of the Mediator complex and further extend the functional similarities between Saccharomyces cerevisiae and metazoan Mediator complexes.

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