Functional Architecture of T7 RNA Polymerase Transcription Complexes

Nayak, Dhananjaya; Guo, Qing; Sousa, Rui

doi:10.1016/j.jmb.2007.05.070

Cited by 9 publications

(9 citation statements)

References 30 publications

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“…Residue 1125 is at the proximal end of the loop and is predicted to be close to the downstream DNA. A nuclease tethered to residue 1125 allows us to test the model's prediction that the DNA downstream of the transcription start site is sharply bent back toward the upstream DNA, as seen in the T7 IC (22)(23)(24)(25). Such an architecture would predict that a nuclease at 1125 would cleave the T-strand around ϩ6 in an IC3.…”

Section: Chemical Nucleases Tethered To the Putative Promoter Recognimentioning

confidence: 99%

A Promoter Recognition Mechanism Common to Yeast Mitochondrial and Phage T7 RNA Polymerases

Nayak

Guo

Sousa

2009

Journal of Biological Chemistry

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Yeast mitochondrial (YMt) and phage T7 RNA polymerases (RNAPs) are two divergent representatives of a large family of single subunit RNAPs that are also found in the mitochondria and chloroplasts of higher eukaryotes, mammalian nuclei, and many other bacteriophage. YMt and phage T7 promoters differ greatly in sequence and length, and the YMt RNAP uses an accessory factor for initiation, whereas T7 RNAP does not. We obtain evidence here that, despite these apparent differences, both the YMt and T7 RNAPs utilize a similar promoter recognition loop to bind their respective promoters. Mutations in this element in YMt RNAP specifically disrupt mitochondrial promoter utilization, and experiments with site-specifically tethered chemical nucleases indicate that this element binds the mitochondrial promoter almost identically to how the promoter recognition loop from the phage RNAP binds its promoter. Sequence comparisons reveal that the other members of the single subunit RNAP family display loops of variable sequence and size at a position corresponding to the YMt and T7 RNAP promoter recognition loops. We speculate that these elements may be involved in promoter recognition in most or all of these enzymes and that this element's structure allows it to accommodate significant sequence and length variation to provide a mechanism for rapid evolution of new promoter specificities in this RNAP family. S. cerevisiae YMt2 and phage T7 RNAPs are both members of the single subunit RNAP family, but their promoter sequences are very different. The T7 promoter is a 21-nucleotide (nt) duplex that includes 4 nt downstream of the transcription start site and 17 nt upstream of ϩ1 (1). The promoters of the closely related T3 phage or the more distantly related Sp6 phage(2) are identical in size to the T7 promoter but differ in sequence. In contrast, the YMt promoter is only 8 nt in length and is therefore similar in size to chloroplast promoters or the mitochondrial promoters of plants and other fungi, which are typically 8 -10 nt in length although highly disparate in sequence (3, 4) (Fig. 1).The mechanisms of promoter recognition by the YMt and T7 RNAP also appear to be distinct. T7 RNAP, like many of the phage polymerases, binds, melts, and initiates transcription as single subunit enzyme without any accessory transcription factor (5). In contrast, the YMt RNAP requires a specific accessory factor (Mtf1 (yeast mitochondrial transcription factor)) to initiate transcription from its promoter (6). This seeming disparity may, however, obscure underlying mechanistic similarity. For example, it was originally suggested (6) that Mtf1 might be homologous and analogous to prokaryotic RNAP -factor, which would mean that Mtf1 contributes directly to promoter recognition, a mechanism distinct from that seen with T7 RNAP, where the polymerase contains all of the promoter recognition elements. However, subsequent work showed that YMt RNAP can initiate transcription without Mtf1 if the promoter is supercoiled or contains a heterduplex ("bubble") ...

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Section: Chemical Nucleases Tethered To the Putative Promoter Recognimentioning

confidence: 99%

A Promoter Recognition Mechanism Common to Yeast Mitochondrial and Phage T7 RNA Polymerases

Nayak

Guo

Sousa

2009

Journal of Biological Chemistry

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“…Mutations in most of the targeted residues had no effects on CJ termination, even when multiple negative charges were introduced into the positively charged region, including residues 407, 412, and 332, that is expected to interact with the duplex DNA upstream of the transcription bubble. 25 The mutants that did affect termination at the CJ element could be grouped into two classes based on their phenotypic effects, the character of the wildtype (WT) side chain, and their location. One class corresponded to mutants in the (mostly aromatic) residues that define the upstream edge of the transcription bubble (Y178, W377, Y385, and D388; Fig.…”

Section: Resultsmentioning

confidence: 99%

“…5a and b). This indicates that the change in the EC structure occurs before it reaches the pause/termination site and most likely coincides with the [16][17][18][19][20], and 207 (lanes [21][22][23][24][25] in ECs halted at + 15 (lanes 2, 7, 12, 17, and 22) or +19 (lanes 3, 8, 13, 18, and 23), or chased for 10 s (lanes 4, 9, 14, 19, and 24) or 10 min (lanes 5, 10, 15, 20, and 25) with a complete NTP mix after stopping the EC at +14 by omission of UTP and CTP from the transcription reaction. The template was HTSCJ66, with the NT strand 32 P-labeled at its 5′-end.…”

Section: Mutations In Residues Near the Upstream Edge Of The Transcrimentioning

confidence: 96%

“…2a). 25 A possible explanation for this result is that, during pausing or termination, the upstream DNA moves away from residues 407 and 412 towards residues 206 and 207. To test this, we compared the cleavage patterns, in normal and CJ-paused ECs, of chemical nucleases tethered to cysteines introduced at residue 407 or 412, or at sites on the N-terminal domain (residues 130, 239, and 207) that could be informative of how the upstream DNA is bound in the EC.…”

Section: Mutations In Residues Near the Upstream Edge Of The Transcrimentioning

confidence: 99%

“…25 Proteolytically nicked RNAP was prepared by incubating purified T7 RNAP with whole Escherichia coli cells (strain HMS174) as described. 38 …”

Section: Rnap Preparationmentioning

confidence: 99%

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Mechanism of T7 RNAP Pausing and Termination at the T7 Concatemer Junction: A Local Change in Transcription Bubble Structure Drives a Large Change in Transcription Complex Architecture

Nayak¹,

Siller²,

Guo³

et al. 2008

Journal of Molecular Biology

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The T7RNA polymerase (RNAP) elongation complex (EC) pauses and is destabilized at a unique 8 nucleotide (nt) sequence found at the junction of the head-to-tail concatemers of T7 genomic DNA generated during T7 DNA replication. The paused EC may recruit the T7 DNA processing machinery, which cleaves the concatemerized DNA within this 8 nt concatemer junction (CJ). Pausing of the EC at the CJ involves structural changes in both the RNAP and transcription bubble. However, these structural changes have not been fully defined, nor is it understood how the CJ sequence itself causes the EC to change its structure, to pause, and to become less stable. Here we use solution and RNAP-tethered chemical nucleases to probe the CJ transcript and changes in the EC structure as the polymerase pauses and terminates at the CJ. Together with extensive mutational scanning of regions of the polymerase that are likely to be involved in recognition of the CJ, we are able to develop a description of the events that occur as the EC transcribes through the CJ and subsequently pauses. In this process, a local change in the structure of the transcription bubble drives a large change in the architecture of the EC. This altered EC structure may then serve as the signal that recruits the processing machinery to the CJ.

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The highly efficient T7 RNA polymerase: A wonder macromolecule in biological realm

Borkotoky

Murali

2018

International Journal of Biological Macromolecules

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Functional Architecture of T7 RNA Polymerase Transcription Complexes

Cited by 9 publications

References 30 publications

A Promoter Recognition Mechanism Common to Yeast Mitochondrial and Phage T7 RNA Polymerases

A Promoter Recognition Mechanism Common to Yeast Mitochondrial and Phage T7 RNA Polymerases

Mechanism of T7 RNAP Pausing and Termination at the T7 Concatemer Junction: A Local Change in Transcription Bubble Structure Drives a Large Change in Transcription Complex Architecture

The highly efficient T7 RNA polymerase: A wonder macromolecule in biological realm

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