A comparison of nucleoside (β-S) triphosphates and nucleoside (γ-S) triphosphates as suitable substrates for measuring transcription initiation in preparations of cell nuclei

Washington, Lynette D.; Stallcup, Michael R.

doi:10.1093/nar/10.24.8311

Cited by 10 publications

(4 citation statements)

References 16 publications

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“…In studies of initiation of RNA synthesis, guanosine 5'-O-(2-thiotriphosphate) (1-S-GTP) or ,B-S-ATP was substituted for GTP or ATP, respectively. The initiated RNA was selected by chromatography on mercuriagarose as described previously (25,35,38).…”

Section: Methodsmentioning

confidence: 99%

Synthesis of U1 RNA in isolated mouse cell nuclei: initiation and 3'-end formation.

Lobo¹,

Marzluff²

1987

Mol. Cell. Biol.

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The transcription of Ul RNA genes was studied in isolated nuclei from mouse myeloma cells. Using a cloned Ulb gene as a probe, we showed that isolated nuclei synthesize both Ulb and Ula RNA. The Ul RNAs were initiated in vitro, as measured by incorporation of adenosine 5'-O-(2-thiotriphosphate) into Ul RNA. There was transcription of the 3'-flanking region but no transcription of regions directly 5' to the Ul genes. In addition to Ul RNAs of the correct length which were released from the nuclei, there were larger RNAs, presumably resulting from transcription into the 3'-flanking region, which were retained in the nuclei. Chase experiments showed that these longer transcripts were not precursors to mature Ul RNA, a finding consistent with the idea that 3'-end formation is coincident with transcription. During the chase, there was maturation of the 3' ends of Ula and Ulb RNAs from slightly longer precursors. In addition to accurate transcription of Ul RNA, there was also synthesis of U2 and U3 RNA. All three of these RNAs were transcribed by RNA polymerase II, as measured by their sensitivity to a-amanitin.The U series of small nuclear RNAs (snRNAs) is one of the most abundant transcripts in mammalian cells. These RNAs are encoded by multiple genes and contain an unusual 5' cap structure, 2,2,7 trimethyl guanosine (3). These RNAs are synthesized by RNA polymerase II, as measured by the sensitivity of their synthesis to low concentrations of aamanitin (8,25,33). An exception is U6 RNA, which lacks the cap structure found at the 5' ends of other U RNAs and is believed to be transcribed by RNA polymerase III (13,32). Some aspects of the synthesis of Ul, U2, and U3 RNAs differ from those of other RNAs synthesized by RNA polymerase II. While cell-free systems have been described which transcribe most other genes accurately, the cell-free systems currently available from mammalian cells do not accurately transcribe the Ul or U2 genes (28, 39). We have previously described the development of both nuclear (25) and DNA-dependent (26) systems from sea urchin embryos which transcribe the sea urchin Ul RNA gene accurately.The promoters of the vertebrate U snRNAs differ from those of other genes transcribed by polymerase II. There is a sequence about 200 nucleotides 5' to the gene which is essential for transcription in vivo (19) and on injection of the genes into Xenopus oocytes (29,33,39). When the genes are used as a template in a DNA-dependent system, there is transcription which initiates about 25 bases downstream from this upstream promoter element (as if it were functioning like a TATAA box in this system) but no transcription which initiates at the proper start site (28). The promoters of the sea urchin U RNA genes are not utilized in Xenopus oocytes (37), unlike the promoters of other sea urchin genes transcribed by RNA polymerase II.The formation of the 3' end of the U snRNAs is unusual in two respects. There is initial formation of a transcript a few nucleotides longer than the mature RNA (6,18,41 (18).Dissecting...

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Section: Methodsmentioning

confidence: 99%

Synthesis of U1 RNA in isolated mouse cell nuclei: initiation and 3'-end formation.

Lobo¹,

Marzluff²

1987

Mol. Cell. Biol.

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“…These might include randomization of the thionucleotide to other nucleotides (4) or the transfer of the thiophosphate to a previously initiated RNA. Transfer of thiophosphate to RNA might occur with -y-S-ATP but does not occur with P-S-ATP (39). Randomization of the label among different nucleotides at the y-position would result in apparent lack of specificity in the initiating nucleotide.…”

Section: Methodsmentioning

confidence: 99%

Synthesis of U1 RNA in isolated nuclei from sea urchin embryos: U1 RNA is initiated at the first nucleotide of the RNA.

Morris¹,

Marzluff²

1985

Mol. Cell. Biol.

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Nuclei from sea urchin blastula embryos synthesize a variety of small RNAs, one of which has identical mobility with sea urchin Ul RNA. This RNA is synthesized by RNA polymerase II and, in a hybridizationselection experiment, was selected by the cloned sea urchin Ul gene. The Ul RNA was initiated with ATP, but not GIP, in isolated nuclei with P-S-and y-S-ribonucleotide triphosphates as substrates. The Ul RNA containing thiophosphate at the 5' end was not capped but accumulated as an uncapped transcript from which the thiophosphate could be removed with calf intestinal phosphatase.

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“…The use of flS-ATP should prevent transfer of the thiol group to in vivo initiated transcripts by RNA kinase activity [10]. Affinity labelling of total endogenous in vitro [ 3 H ] transcripts was reduced when flS-ATP was used instead of 7S-ATP, but was still above the non-specific background obtained with ATP (Table 1).…”

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confidence: 99%

“…However, post-transcriptional affinity labelling of in vivo initiated RNA might be a major problem, and transfer of the 7-thiol group to exogenous RNA has been described in isolated nuclei [3,10]. Such post-transcriptional affinity labelling can occur upon combined phosphatase/ RNA kinase or RNase/RNA kinase activity.…”

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confidence: 99%

Post-transcriptional transfer of ?-thio affinity label to RNA in isolated parsley nuclei

Schweizer

Hahlbrock

1993

Plant Mol Biol

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As an alternative to soluble plant transcription systems, we examined the reinitiation capacity of isolated parsley nuclei. Nuclear chalcone synthase in vitro transcripts were affinity-labelled with gamma-thio-ATP, gamma-thio-GTP or beta-thio-ATP, and purified by chromatography on a mercury Sepharose affinity column. Primer extension and subsequent PCR amplification of these in vitro transcripts revealed gamma-thio-ATP-dependent, but no beta-thio-ATP-dependent, signals, although affinity labelling of overall in vitro transcripts still occurred with beta-thio-ATP. We conclude that the described plant nuclei reinitiated transcription non-specifically and that post-transcriptional transfer of the gamma-thio affinity label severely interfered with the detection of reinitiated transcripts.

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A comparison of nucleoside (β-S) triphosphates and nucleoside (γ-S) triphosphates as suitable substrates for measuring transcription initiation in preparations of cell nuclei

Cited by 10 publications

References 16 publications

Synthesis of U1 RNA in isolated mouse cell nuclei: initiation and 3'-end formation.

Synthesis of U1 RNA in isolated mouse cell nuclei: initiation and 3'-end formation.

Synthesis of U1 RNA in isolated nuclei from sea urchin embryos: U1 RNA is initiated at the first nucleotide of the RNA.

Post-transcriptional transfer of ?-thio affinity label to RNA in isolated parsley nuclei

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