Direct Versus Limited-step Reconstitution Reveals Key Features of an RNA Hairpin-stabilized Paused Transcription Complex

Scotty Kyzer,Murali Palangat,Robert Landick,Kook Sun Ha

doi:10.1074/jbc.m701483200

Scotty Kyzer, Murali Palangat + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m701483200

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Abstract

We have identified minimal nucleic acid scaffolds capable of reconstituting hairpin-stabilized paused transcription complexes when incubated with RNAP either directly or in a limited step reconstitution assay. Direct reconstitution was achieved using a 29-nucleotide (nt) RNA whose 3'-proximal 9-10 nt pair to template DNA within an 11-nt noncomplementary bubble of a 39-bp duplex DNA; the 5'-proximal 18 nt of RNA forms the his pause RNA hairpin. Limited-step reconstitution was achieved on the same DNAs using a 27-nt RNA that can be 3'-labeled during reconstitution and then extended 2 nt past the pause site to assay transcriptional pausing. Paused complexes formed by either method recapitulated key features of a promoter-initiated, hairpin-stabilized paused complex, including a slow rate of pause escape, resistance to transcript cleavage and pyrophosphorolysis, and enhancement of pausing by the elongation factor NusA. These findings establish that RNA upstream from the pause hairpin and pyrophosphate are not essential for pausing and for NusA action. Reconstitution of the his paused transcription complex provides a valuable tool for future studies of protein-nucleic interactions involved in transcriptional pausing.

Highlights

Of Escherichia coli, RNAP pauses until a translating ribosome is able to direct transcription readthrough or termination in response to the level of charged histidyl-tRNA [1, 2]
paused transcription complex (PTC) can be directly reconstituted at the pause site using nucleic acid scaffolds that contain the pause hairpin but no upstream RNA, and these reconstituted PTCs (rPTCs) behave kinetically like PTCs initiated from a promoter
NusA increases the lifetime of his rPTCs lacking upstream RNA to about the same extent at promoter-initiated PTCs containing a pause RNA

Summary

Introduction

Of Escherichia coli, RNAP pauses until a translating ribosome is able to direct transcription readthrough or termination in response to the level of charged histidyl-tRNA [1, 2]. ␴70 with RNAP shortly after promoter escape) [12] The his pause (Class I) is stabilized by the interaction of a 5-bp stem, 8-nt loop pause RNA hairpin that forms 11 nt from the paused transcript 3Ј end and contributes a factor of ϳ10 of an overall 100 –1000-fold decrease in the nucleotide addition rate. The duration of the his pause can be increased ϳ2– 4-fold by the elongation factor NusA, which interacts with the pause hairpin and with RNAP [7, 9] The his PTC remains pretranslocated but is resistant to pyrophosphorolysis, which ordinarily occurs rapidly in pretranslocated complexes [8]. This suggests that the active site of the his PTC is altered such that catalysis is blocked

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