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Factor-dependent Release of Nascent RNA by Ternary Complexes of Vaccinia RNA Polymerase
Factor-dependent transcription termination during synthesis of vaccinia early mRNAs occurs at heterogeneous sites downstream of a UUUUUNU signal in the nascent transcript. The choice of termination site is flexible and is determined by a kinetic balance between nascent chain elongation and the trans...
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Published in: | The Journal of biological chemistry 1996-08, Vol.271 (32), p.19556-19562 |
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Main Authors: | , , |
Format: | Article |
Language: | English |
Subjects: | |
Citations: | Items that this one cites Items that cite this one |
Online Access: | Get full text |
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Summary: | Factor-dependent transcription termination during synthesis of vaccinia early mRNAs occurs at heterogeneous sites downstream
of a UUUUUNU signal in the nascent transcript. The choice of termination site is flexible and is determined by a kinetic balance
between nascent chain elongation and the transmission of the RNA signal to the polymerase. To eliminate ongoing elongation
as a variable, we have established a system to study transcript release by purified ternary complexes halted at a defined
template position 50-nucleotides 3â² of the first U residue of the termination signal. Release of the nascent RNA depends on
the vaccinia termination factor (VTF) and an ATP cofactor. Transcript release is blocked by BrUMP substitution within the
termination signal of the nascent RNA. In these respects, the release reaction faithfully mimics the properties of the termination
event. We demonstrate that ternary complexes are refractory to VTF-mediated transcript release when the first U of the UUUUUNU
signal is situated 20 nucleotides from the growing point of the nascent chain. Ribonuclease footprinting of the arrested ternary
complexes defines a nascent RNA binding site on the polymerase elongation complex that encompasses a 16-21 nucleotide RNA
segment extending proximally from the 3â² end of the chain. We surmise that access of VTF to the signal sequence is prevented
when UUUUUNU is bound within the nascent RNA binding site. Hence, physical not kinetic constraints determine the minimal distance
between the signal and potential sites of 3â² end formation. |
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ISSN: | 0021-9258 1083-351X |
DOI: | 10.1074/jbc.271.32.19556 |