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

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.

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

10.1101/2020.06.30.179606 ◽

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.

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An Evolutionary/Biochemical Connection between Promoter- and Primer-Dependent Polymerases Revealed by Systematic Evolution of Ligands by Exponential Enrichment

Journal of Bacteriology ◽

10.1128/jb.00579-17 ◽

2018 ◽

Vol 200 (7) ◽

Author(s):

Katherine J. Fenstermacher ◽

Vasudevan Achuthan ◽

Thomas D. Schneider ◽

Jeffrey J. DeStefano

Keyword(s):

Core Promoter ◽

Dna Polymerases ◽

Promoter Sequence ◽

Rna Polymerases ◽

Sequence Specificity ◽

High Affinity ◽

Phage T7 ◽

Systematic Evolution ◽

Exponential Enrichment ◽

Dna Primers

ABSTRACTDNA polymerases (DNAPs) recognize 3′ recessed termini on duplex DNA and carry out nucleotide catalysis. Unlike promoter-specific RNA polymerases (RNAPs), no sequence specificity is required for binding or initiation of catalysis. Despite this, previous results indicate that viral reverse transcriptases bind much more tightly to DNA primers that mimic the polypurine tract. In the current report, primer sequences that bind with high affinity toTaqand Klenow polymerases were identified using a modified systematic evolution of ligands by exponential enrichment (SELEX) approach. TwoTaq-specific primers that bound ∼10 (Taq1) and over 100 (Taq2) times more stably than controls toTaqwere identified. TaqI contained 8 nucleotides (5′-CACTAAAG-3′) that matched the phage T3 RNAP “core” promoter. Both primers dramatically outcompeted primers with similar binding thermodynamics in PCRs. Similarly, exonuclease−Klenow polymerase also selected a high-affinity primer that contained a related core promoter sequence from phage T7 RNAP (5′-ACTATAG-3′). For bothTaqand Klenow, even small modifications to the sequence resulted in large losses in binding affinity, suggesting that binding was highly sequence specific. The results are discussed in the context of possible effects on multiprimer (multiplex) PCR assays, molecular information theory, and the evolution of RNAPs and DNAPs.IMPORTANCEThis work further demonstrates that primer-dependent DNA polymerases can have strong sequence biases leading to dramatically tighter binding to specific sequences. These may be related to biological function or be a consequence of the structural architecture of the enzyme. New sequence specificity forTaqand Klenow polymerases were uncovered, and among them were sequences that contained the core promoter elements from T3 and T7 phage RNA polymerase promoters. This suggests the intriguing possibility that phage RNA polymerases exploited intrinsic binding affinities of ancestral DNA polymerases to develop their promoters. Conversely, DNA polymerases could have evolved from related RNA polymerases and retained the intrinsic binding preference despite there being no clear function for such a preference in DNA biology.

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