scholarly journals Conservation of Promoter Melting Mechanisms in Divergent Regions of the Single-Subunit RNA Polymerases

Biochemistry ◽  
2012 ◽  
Vol 51 (18) ◽  
pp. 3901-3910 ◽  
Author(s):  
Gilberto Velazquez ◽  
Qing Guo ◽  
Liping Wang ◽  
Luis G. Brieba ◽  
Rui Sousa
Keyword(s):  
Rna Polymerases ◽  
Promoter Melting ◽  
Single Subunit

scholarly journals Transcriptional fidelities of human mitochondrial POLRMT, yeast mitochondrial Rpo41, and phage T7 single-subunit RNA polymerases

2017 ◽  
Vol 292 (44) ◽  
pp. 18145-18160 ◽  
Author(s):  
Shemaila Sultana ◽  
Mihai Solotchi ◽  
Aparna Ramachandran ◽  
Smita S. Patel
Keyword(s):  
Rna Polymerases ◽  
Phage T7 ◽  
Single Subunit

On the Evolution of the Single-Subunit RNA Polymerases

1997 ◽  
Vol 45 (6) ◽  
pp. 671-681 ◽  
Author(s):  
Nicolas Cermakian ◽  
Tatsuya M. Ikeda ◽  
Pedro Miramontes ◽  
B. Franz Lang ◽  
Michael W. Gray ◽  
...  
Keyword(s):  
Rna Polymerases ◽  
Single Subunit

scholarly journals The Length of Promoter Sequence Affects The De Novo Initiation By T7 RNA Polymerase in Vitro: New Insights Into The Evolution of Promoters For Single Subunit RNA Polymerases

2019 ◽  
Author(s):  
Ramesh Padmanabhan ◽  
Dennis Miller
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
De Novo ◽  
Promoter Sequence ◽  
Rna Polymerases ◽  
T7 Rna Polymerase ◽  
Promoter Sequences ◽  
Promoter Recognition ◽  
Single Subunit

1.1AbstractRNA polymerases (RNAPs) differ from other polymerases in that they can bind promoter sequences and initiate de novo transcription. Promoter recognition requires the presence of specific DNA binding domains in the polymerase. The structure and mechanistic aspects of transcription by the bacteriophage T7 RNA polymerase (T7 RNAP) are well characterized. This single subunit RNAP belongs to the family of RNAPs which also includes the T3, SP6 and mitochondrial RNAPs. High specificity for its promoter, the requirement of no additional transcription factors, and high fidelity of initiation from a specific site in the promoter makes it the polymerase of choice to study the mechanistic aspects of transcription. The structure and function of the catalytic domains of this family of polymerases are highly conserved suggesting a common mechanism underlying transcription. Although the two groups of single subunit RNAPs, mitochondrial and bacteriophage, have remarkable structural conservation, they recognize quite dissimilar promoters. Specifically, the bacteriophage promoters recognize a 23 nucleotide promoter extending from −17 to + 6 nucleotides relative to the site of transcription initiation, while the well characterized promoter recognized by the yeast mitochondrial RNAP is nine nucleotides in length extending from −8 to +1 relative to the site of transcription initiation. Promoters recognized by the bacteriophage RNAPs are also well characterized with distinct functional domains involved in promoter recognition and transcription initiation. Thorough mutational studies have been conducted by altering individual base-pairs within these domains. Here we describe experiments to determine whether the prototype bacteriophage RNAP is able to recognize and initiate at truncated promoters similar to mitochondrial promoters. Using an in vitro oligonucleotide transcriptional system, we have assayed transcription initiation activity by T7 RNAP. When a complete or almost complete (20 to 16 nucleotide) double stranded T7 RNAP promoter sequence is present, small RNA’s are produced through template-independent and promoter-dependent stuttering corresponding to abortive initiation, and this effect was lost with a scrambled promoter sequence. When partial double stranded promoter sequences (10 to 12 nucleotides) are supplied, template dependent de novo initiation of RNA occurs at a site different from the canonical +1-initiation site. The site of transcription initiation is determined by a recessed 3’ end based paired to the template strand of DNA rather than relative to the partial promoter sequence. Understanding the mechanism underlying this observation helps us to understand the role of the elements in the T7 promoter, and provides insights into the promoter evolution of the single-subunit RNAPs.

scholarly journals The mechanism of the nucleo-sugar selection by multi-subunit RNA polymerases

2020 ◽  
Author(s):  
Janne J. Mäkinen ◽  
Yeonoh Shin ◽  
Eeva Vieras ◽  
Pasi Virta ◽  
Mikko Metsä-Ketelä ◽  
...  
Keyword(s):  
Dna Polymerases ◽  
Rna Polymerases ◽  
Selective Inhibitors ◽  
Evolutionary Origins ◽  
Active Selection ◽  
Selection Strategies ◽  
Single Subunit

AbstractRNA polymerases (RNAPs) synthesize RNA from NTPs, whereas DNA polymerases synthesize DNA from 2’dNTPs. DNA polymerases select against NTPs by using steric gates to exclude the 2’ OH, but RNAPs have to employ alternative selection strategies. In single-subunit RNAPs, a conserved Tyr residue discriminates against 2’dNTPs, whereas selectivity mechanisms of multi-subunit RNAPs remain hitherto unknown. Here we show that a conserved Arg residue uses a two-pronged strategy to select against 2’dNTPs in multi-subunit RNAPs. The conserved Arg interacts with the 2’OH group to promote NTP binding, but selectively inhibits incorporation of 2’dNTPs by interacting with their 3’OH group to favor the catalytically-inert 2’-endo conformation of the deoxyribose moiety. This deformative action is an elegant example of an active selection against a substrate that is a substructure of the correct substrate. Our findings provide important insights into the evolutionary origins of biopolymers and the design of selective inhibitors of viral RNAPs.

scholarly journals The mechanism of the nucleo-sugar selection by multi-subunit RNA polymerases

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Janne J. Mäkinen ◽  
Yeonoh Shin ◽  
Eeva Vieras ◽  
Pasi Virta ◽  
Mikko Metsä-Ketelä ◽  
...  
Keyword(s):  
Dna Polymerases ◽  
Rna Polymerases ◽  
Selective Inhibitors ◽  
Evolutionary Origins ◽  
Active Selection ◽  
Selection Strategies ◽  
Single Subunit

AbstractRNA polymerases (RNAPs) synthesize RNA from NTPs, whereas DNA polymerases synthesize DNA from 2′dNTPs. DNA polymerases select against NTPs by using steric gates to exclude the 2′OH, but RNAPs have to employ alternative selection strategies. In single-subunit RNAPs, a conserved Tyr residue discriminates against 2′dNTPs, whereas selectivity mechanisms of multi-subunit RNAPs remain hitherto unknown. Here, we show that a conserved Arg residue uses a two-pronged strategy to select against 2′dNTPs in multi-subunit RNAPs. The conserved Arg interacts with the 2′OH group to promote NTP binding, but selectively inhibits incorporation of 2′dNTPs by interacting with their 3′OH group to favor the catalytically-inert 2′-endo conformation of the deoxyribose moiety. This deformative action is an elegant example of an active selection against a substrate that is a substructure of the correct substrate. Our findings provide important insights into the evolutionary origins of biopolymers and the design of selective inhibitors of viral RNAPs.

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