RNA Polymerase Active Center: The Molecular Engine of Transcription

Nudler, Evgeny

doi:10.1146/annurev.biochem.76.052705.164655

Cited by 137 publications

(120 citation statements)

References 152 publications

(384 reference statements)

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“…For DHBV/Hε, we did not detect accumulation of specific new mutations during repeated in vivo passages, and for DHBV/S1, the same four mutations emerged in separate ducks yet remained stable thereafter. This may appear surprising given that progeny rcDNA formation involves two error-prone copying processes, i.e., transcription of pgRNA by host RNA polymerase II, with an estimated error rate of around 10 Ϫ5 per site (41), and reverse transcription of pgRNA by the viral polymerase (involving synthesis of two new DNA strands). Comparability of many published "mutation rates" suffers from different experimental setups and experimental imperfections (49) and the promiscuous use of different definitions of the term; useful distinctions are between the error rate of a polymerase, the mutation rate which accounts for additional mutations introduced by other events, and the substitution rate, which refers to those mutations that become fixed in the progeny, a complex product of mutation rate, generation time, population size, and fitness (15).…”

Section: Discussionmentioning

confidence: 99%

A High Level of Mutation Tolerance in the Multifunctional Sequence Encoding the RNA Encapsidation Signal of an Avian Hepatitis B Virus and Slow Evolution Rate Revealed by In Vivo Infection

Schmid

Rösler

Nassal

2011

J Virol

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In all hepadnaviruses, protein-primed reverse transcription of the pregenomic RNA (pgRNA) is initiated by binding of the viral polymerase, P protein, to the RNA element. Universally, consists of a lower stem and an upper stem, separated by a bulge, and an apical loop. Complex formation triggers pgRNA encapsidation and the -templated synthesis of a DNA oligonucleotide (priming) that serves to generate minus-strand DNA. In vitro systems for duck hepatitis B virus (DHBV) yielded important insights into the priming mechanism, yet their relevance in infection is largely unexplored. Moreover, additional functions encoded in the DHBV (D) sequence could affect in vivo fitness. We therefore assessed the in vivo performances of five recombinant DHBVs bearing multiple mutations in the upper D stem. Three variants with only modestly reduced in vitro replication competence established chronic infection in ducks. From one variant but not another, three adapted new variants emerged upon passaging, as demonstrated by increased relative fitness in coinfections with wild-type DHBV. All three showed enhanced priming and replication competence in vitro, and in one, DHBV e antigen (DHBeAg) production was restored. Pronounced impacts on other D functions were not detected; however, gradual, synergistic contributions to overall performance are suggested by the fact of none of the variants reaching the in vivo fitness of wild-type virus. These data shed more light on the P-D interaction, define important criteria for the design of future in vivo evolution experiments, and suggest that the upper D stem sequences provided an evolutionary playground for DHBV to optimize in vivo fitness.Hepadnaviruses are small enveloped hepatotropic DNA viruses that replicate through protein-primed reverse transcription (for reviews, see references 8 and 38). Human hepatitis B virus (HBV) and duck HBV (DHBV) are the respective prototypes of the mammalian and avian HBVs, which share a similar genome organization and replication strategy (Fig. 1A). Their ϳ3-kb genomes encode a single core protein, three or two (avian viruses) envelope proteins, an unusual reverse transcriptase (P protein), and, only in the mammalian viruses, the poorly understood X protein. Virions contain the genome as a relaxed circular DNA (rcDNA), which upon infection is converted into nuclear covalently closed circular DNA (cccDNA), the template for all viral transcripts. In addition to the subgenomic RNAs (sgRNAs), two types of greater-than-genomelength RNAs are produced. The precore RNAs serve for translation of the precursors of the secretory hepatitis B virus e antigens (HBeAg and DHBeAg, respectively), which may modulate the host's immune response but are nonessential. The pregenomic RNAs (pgRNAs), by contrast, are vital as mRNAs for core and P protein and by constituting the obligate template for new DNA genomes.Reverse transcription begins with specific packaging of pgRNA into newly forming capsids, triggered by binding of P protein to the 5Ј-proximal ε (Dε for DHBV) stem-loop (...

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Section: Discussionmentioning

confidence: 99%

A High Level of Mutation Tolerance in the Multifunctional Sequence Encoding the RNA Encapsidation Signal of an Avian Hepatitis B Virus and Slow Evolution Rate Revealed by In Vivo Infection

Schmid

Rösler

Nassal

2011

J Virol

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“…The binding of proteins to the transcript could prevent the translocation of RNAPII by preventing the movement of the transcript in and out of the RNA exit channel. There is evidence that the binding of proteins to the emerging transcript can favor elongation by disfavoring backward translocation of the polymerase (Reeder and Hawley 1996;Roberts et al 2008;Nudler 2009;Proshkin et al 2010). We propose that Ccr4-Not stimulates elongation by promoting realignment of the 39 end of the transcript in the active site by trapping RNAPII during its forward excursions along the template by binding to the transcript and preventing backward transitions.…”

Section: Mechanism For Ccr4-not Function During Elongationmentioning

confidence: 92%

“…Forward translocation of RNAPII occurs via Brownian motion, and stalled ECs are believed to undergo excursions in the forward and reverse directions Nudler 2009). Arrested RNAPII can move along the template in the forward and backward directions, causing the threading of the transcript through the RNA exit channel.…”

Section: Mechanism For Ccr4-not Function During Elongationmentioning

confidence: 99%

The multifunctional Ccr4–Not complex directly promotes transcription elongation

Kruk¹,

Dutta²,

Fu³

et al. 2011

Genes Dev.

156

228

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The Ccr4-Not complex has been implicated in the control of multiple steps of mRNA metabolism; however, its functions in transcription remain ambiguous. The discovery that Ccr4/Pop2 is the major cytoplasmic mRNA deadenylase and the detection of Not proteins within mRNA processing bodies have raised questions about the roles of the Ccr4-Not complex in transcription. Here we firmly establish Ccr4-Not as a positive elongation factor for RNA polymerase II (RNAPII). The Ccr4-Not complex is targeted to the coding region of genes in a transcription-dependent manner similar to RNAPII and promotes elongation in vivo. Furthermore, Ccr4-Not interacts directly with elongating RNAPII complexes and stimulates transcription elongation of arrested polymerase in vitro. Ccr4-Not can reactivate backtracked RNAPII using a mechanism different from that of the well-characterized elongation factor TFIIS. While not essential for its interaction with elongation complexes, Ccr4-Not interacts with the emerging transcript and promotes elongation in a manner dependent on transcript length, although this interaction is not required for it to bind RNAPII. Our comprehensive analysis shows that Ccr4-Not directly regulates transcription, and suggests it does so by promoting the resumption of elongation of arrested RNAPII when it encounters transcriptional blocks in vivo.

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“…Each extension step must include formation of a Watson-Crick base pair, and a transphosphorylation step, in which a leaving group is replaced by a nucleophile from the primer (1). The same type of reaction occurs during transcription, except that an RNA strand is formed (2). Copying lies at the heart of our understanding of genetics, but this reaction with its multiple substrates continues to be enigmatic (3).…”

mentioning

confidence: 99%

Templating efficiency of naked DNA

Kervio

Hochgesand

Steiner

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

111

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Template-directed synthesis of complementary strands is pivotal for life. Nature employs polymerases for this reaction, leaving the ability of DNA itself to direct the incorporation of individual nucleotides at the end of a growing primer difficult to assess. Using 64 sequences, we now find that any of the four nucleobases, in combination with any neighboring residue, support enzyme-free primer extension when primer and mononucleotide are sufficiently reactive, with ≥93% primer extension for all sequences. Between the 64 possible base triplets, the rate of extension for the poorest template, CAG, with A as templating base, and the most efficient template, TCT, with C as templating base, differs by less than two orders of magnitude. Further, primer extension with a balanced mixture of monomers shows ≥72% of the correct extension product in all cases, and ≥90% incorporation of the correct base for 46 out of 64 triplets in the presence of a downstream-binding strand. A mechanism is proposed with a binding equilibrium for the monomer, deprotonation of the primer, and two chemical steps, the first of which is most strongly modulated by the sequence. Overall, rates show a surprisingly smooth reactivity landscape, with similar incorporation on strongly and weakly templating sequences. These results help to clarify the substrate contribution to copying, as found in polymerase-catalyzed replication, and show an important feature of DNA as genetic material.

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RNA Polymerase Active Center: The Molecular Engine of Transcription

Cited by 137 publications

References 152 publications

A High Level of Mutation Tolerance in the Multifunctional Sequence Encoding the RNA Encapsidation Signal of an Avian Hepatitis B Virus and Slow Evolution Rate Revealed by In Vivo Infection

A High Level of Mutation Tolerance in the Multifunctional Sequence Encoding the RNA Encapsidation Signal of an Avian Hepatitis B Virus and Slow Evolution Rate Revealed by In Vivo Infection

The multifunctional Ccr4–Not complex directly promotes transcription elongation

Templating efficiency of naked DNA

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