The RNA Polymerase of Influenza Virus, Bound to the 5′ End of Virion RNA, Acts in cis To Polyadenylate mRNA

ABSTRACT We previously demonstrated, by limited mutagenesis, that conserved sequence elements within the 5′ end of influenza virus virion RNA (vRNA) are required for the polyadenylation of mRNA in vitro. To further characterize the nucleotide residues at the 5′ end of vRNA which might be involved in polyadenylation, a complete set of short and long model vRNA-like templates with mutations at nucleotides 1′ to 13′ (prime notation denotes numbering from the 5′ end) of vRNA were synthesized and transcribed in vitro. The products were assayed for mRNA production with both reverse transcription-PCR and [α-32P]ATP incorporation assays. Results from these independent assays showed that vRNA templates with point mutations at positions 2′, 3′, 7′ to 9′, and 11′ to 13′ synthesized polyadenylated transcripts inefficiently compared with those with mutations at positions 1′, 4′ to 6′, and 10′. Positions 2′, 3′, 7′ to 9′, and 11′ are known to be involved in RNA polymerase binding. Furthermore, residues at positions 11′ to 13′ are known to be involved in base pairing between the 3′ and 5′ ends of vRNA. These findings demonstrate that the RNA polymerase has to bind to the 5′ end of the template vRNA, which must then interact with the 3′ end of the same template for polyadenylation to occur. These results support a model in which acis-acting RNA polymerase is required for the polyadenylation of influenza virus.

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Multiple Regions on the Escherichia coliHeat Shock Transcription Factor ς32 Determine Core RNA Polymerase Binding Specificity

Journal of Bacteriology ◽

10.1128/jb.180.5.1095-1102.1998 ◽

1998 ◽

Vol 180 (5) ◽

pp. 1095-1102 ◽

Cited By ~ 41

Author(s):

Daniel M. Joo ◽

Audrey Nolte ◽

Richard Calendar ◽

Yan Ning Zhou ◽

Ding Jun Jin

Keyword(s):

Rna Polymerase ◽

Point Mutations ◽

In Vitro Transcription ◽

Binding Activity ◽

Sigma Factors ◽

Class Iii ◽

Multiple Regions ◽

Polymerase Binding ◽

Rnap Binding

ABSTRACT We have analyzed the core RNA polymerase (RNAP) binding activity of the purified products of nine defective alleles of the rpoHgene, which encodes ς32 in Escherichia coli. All mutations studied here lie outside of the putative core RNAP binding regions 2.1 and 2.2. Based on the estimatedKs s for the mutant sigma and core RNAP interaction determined by in vitro transcription and by glycerol gradient sedimentation, we have divided the mutants into three classes. The class III mutants showed greatly decreased affinity for core RNAP, whereas the class II mutants’ effect on core RNAP interaction was only clearly seen in the presence of ς70 competitor. The class I mutant behaved nearly identically to the wild type in core RNAP binding. Two point mutations in class III altered residues that were distant from one another. One was found in conserved region 4.2, and the other was in a region conserved only among heat shock sigma factors. These data suggest that there is more than one core RNAP binding region in ς32 and that differences in contact sites probably exist among sigma factors.

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Faculty Opinions recommendation of In vitro transcription profiling of the σS subunit of bacterial RNA polymerase: re-definition of the σS regulon and identification of σS-specific promoter sequence elements.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.12662957.13913055 ◽

2011 ◽

Author(s):

Herb Schellhorn

Keyword(s):

Rna Polymerase ◽

Promoter Sequence ◽

In Vitro Transcription ◽

Transcription Profiling ◽

Bacterial Rna ◽

Sequence Elements ◽

Definition Of

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Characterization of an enhanced antigenic change in the pandemic 2009 H1N1 influenza virus haemagglutinin

Journal of General Virology ◽

10.1099/vir.0.061598-0 ◽

2014 ◽

Vol 95 (5) ◽

pp. 1033-1042 ◽

Cited By ~ 5

Author(s):

Blanca García-Barreno ◽

Teresa Delgado ◽

Sonia Benito ◽

Inmaculada Casas ◽

Francisco Pozo ◽

...

Keyword(s):

Influenza Virus ◽

Amino Acid ◽

Influenza A ◽

Point Mutations ◽

Antigenic Site ◽

Single Amino Acid ◽

Antigenic Structure ◽

Human Antibody ◽

2009 H1n1

Murine hybridomas producing neutralizing mAbs specific to the pandemic influenza virus A/California/07/2009 haemagglutinin (HA) were isolated. These antibodies recognized at least two different but overlapping new epitopes that were conserved in the HA of most Spanish pandemic isolates. However, one of these isolates (A/Extremadura/RR6530/2010) lacked reactivity with the mAbs and carried two unique mutations in the HA head (S88Y and K136N) that were required simultaneously to eliminate reactivity with the murine antibodies. This unusual requirement directly illustrates the phenomenon of enhanced antigenic change proposed previously for the accumulation of simultaneous amino acid substitutions at antigenic sites of the influenza A virus HA during virus evolution (Shih et al., Proc Natl Acad Sci USA, 104 , 6283–6288, 2007). The changes found in the A/Extremadura/RR6530/2010 HA were not found in escape mutants selected in vitro with one of the mAbs, which contained instead nearby single amino acid changes in the HA head. Thus, either single or double point mutations may similarly alter epitopes of the new antigenic site identified in this work in the 2009 H1N1 pandemic virus HA. Moreover, this site is relevant for the human antibody response, as shown by competition of mAbs and human post-infection sera for virus binding. The results are discussed in the context of the HA antigenic structure and challenges posed for identification of sequence changes with possible antigenic impact during virus surveillance.

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Role of the 3′ tRNA-Like Structure in Tobacco Mosaic Virus Minus-Strand RNA Synthesis by the Viral RNA-Dependent RNA Polymerase In Vitro

Journal of Virology ◽

10.1128/jvi.74.24.11671-11680.2000 ◽

2000 ◽

Vol 74 (24) ◽

pp. 11671-11680 ◽

Cited By ~ 49

Author(s):

T. A. M. Osman ◽

C. L. Hemenway ◽

K. W. Buck

Keyword(s):

Rna Polymerase ◽

Tobacco Mosaic Virus ◽

Mosaic Virus ◽

Untranslated Region ◽

Rna Synthesis ◽

Central Core ◽

Minus Strand ◽

Stem Loop ◽

Polymerase Binding

ABSTRACT A template-dependent RNA polymerase has been used to determine the sequence elements in the 3′ untranslated region of tobacco mosaic virus RNA that are required for promotion of minus-strand RNA synthesis and binding to the RNA polymerase in vitro. Regions which were important for minus-strand synthesis were domain D1, which is equivalent to a tRNA acceptor arm; domain D2, which is similar to a tRNA anticodon arm; an upstream domain, D3; and a central core, C, which connects domains D1, D2, and D3 and determines their relative orientations. Mutational analysis of the 3′-terminal 4 nucleotides of domain D1 indicated the importance of the 3′-terminal CA sequence for minus-strand synthesis, with the sequence CCCA or GGCA giving the highest transcriptional efficiency. Several double-helical regions, but not their sequences, which are essential for forming pseudoknot and/or stem-loop structures in domains D1, D2, and D3 and the central core, C, were shown to be required for high template efficiency. Also important were a bulge sequence in the D2 stem-loop and, to a lesser extent, a loop sequence in a hairpin structure in domain D1. The sequence of the 3′ untranslated region upstream of domain D3 was not required for minus-strand synthesis. Template-RNA polymerase binding competition experiments showed that the highest-affinity RNA polymerase binding element region lay within a region comprising domain D2 and the central core, C, but domains D1 and D3 also bound to the RNA polymerase with lower affinity.

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Transcription termination in vitro by bacteriophage T7 RNA polymerase. The role of sequence elements within and surrounding a rho-independent transcription terminator.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)41775-9 ◽

1992 ◽

Vol 267 (27) ◽

pp. 19306-19312 ◽

Cited By ~ 1

Author(s):

S.T. Jeng ◽

J.F. Gardner ◽

R.I. Gumport

Keyword(s):

Rna Polymerase ◽

Transcription Termination ◽

T7 Rna Polymerase ◽

Bacteriophage T7 ◽

Transcription Terminator ◽

Sequence Elements

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Mutational analysis of centromere DNA from chromosome VI of Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.8.6.2523-2535.1988 ◽

1988 ◽

Vol 8 (6) ◽

pp. 2523-2535

Author(s):

J H Hegemann ◽

J H Shero ◽

G Cottarel ◽

P Philippsen ◽

P Hieter

Keyword(s):

Saccharomyces Cerevisiae ◽

Mitotic Chromosome ◽

Mutational Analysis ◽

Point Mutations ◽

Vector System ◽

Chromosome Loss ◽

Wild Type ◽

Sequence Elements ◽

Chromosome Fragments

Saccharomyces cerevisiae centromeres have a characteristic 120-base-pair region consisting of three distinct centromere DNA sequence elements (CDEI, CDEII, and CDEIII). We have generated a series of 26 CEN mutations in vitro (including 22 point mutations, 3 insertions, and 1 deletion) and tested their effects on mitotic chromosome segregation by using a new vector system. The yeast transformation vector pYCF5 was constructed to introduce wild-type and mutant CEN DNAs onto large, linear chromosome fragments which are mitotically stable and nonessential. Six point mutations in CDEI show increased rates of chromosome loss events per cell division of 2- to 10-fold. Twenty mutations in CDEIII exhibit chromosome loss rates that vary from wild type (10(-4)) to nonfunctional (greater than 10(-1)). These results directly identify nucleotides within CDEI and CDEIII that are required for the specification of a functional centromere and show that the degree of conservation of an individual base does not necessarily reflect its importance in mitotic CEN function.

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Structure-function comparisons of (p)ppApp vs (p)ppGpp for Escherichia coli RNA polymerase binding sites and for rrnB P1 promoter regulatory responses in vitro

Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms ◽

10.1016/j.bbagrm.2018.07.005 ◽

2018 ◽

Vol 1861 (8) ◽

pp. 731-742 ◽

Cited By ~ 7

Author(s):

Bożena Bruhn-Olszewska ◽

Vadim Molodtsov ◽

Michał Sobala ◽

Maciej Dylewski ◽

Katsuhiko S. Murakami ◽

...

Keyword(s):

Escherichia Coli ◽

Rna Polymerase ◽

Structure Function ◽

Binding Sites ◽

Polymerase Binding

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Point Mutations in Yeast CBF5 Can Abolish In Vivo Pseudouridylation of rRNA

Molecular and Cellular Biology ◽

10.1128/mcb.19.11.7461 ◽

1999 ◽

Vol 19 (11) ◽

pp. 7461-7472 ◽

Cited By ~ 133

Author(s):

Yeganeh Zebarjadian ◽

Tom King ◽

Maurille J. Fournier ◽

Louise Clarke ◽

John Carbon

Keyword(s):

Catalytic Domain ◽

Sequence Similarity ◽

Point Mutations ◽

Rrna Synthesis ◽

Temperature Sensitive ◽

In Vitro Mutagenesis ◽

Sequence Motifs ◽

Conserved Sequence

ABSTRACT In budding yeast (Saccharomyces cerevisiae), the majority of box H/ACA small nucleolar RNPs (snoRNPs) have been shown to direct site-specific pseudouridylation of rRNA. Among the known protein components of H/ACA snoRNPs, the essential nucleolar protein Cbf5p is the most likely pseudouridine (Ψ) synthase. Cbf5p has considerable sequence similarity to Escherichia coli TruBp, a known Ψ synthase, and shares the “KP” and “XLD” conserved sequence motifs found in the catalytic domains of three distinct families of known and putative Ψ synthases. To gain additional evidence on the role of Cbf5p in rRNA biosynthesis, we have used in vitro mutagenesis techniques to introduce various alanine substitutions into the putative Ψ synthase domain of Cbf5p. Yeast strains expressing these mutatedcbf5 genes in a cbf5Δ null background are viable at 25°C but display pronounced cold- and heat-sensitive growth phenotypes. Most of the mutants contain reduced levels of Ψ in rRNA at extreme temperatures. Substitution of alanine for an aspartic acid residue in the conserved XLD motif of Cbf5p (mutantcbf5D95A) abolishes in vivo pseudouridylation of rRNA. Some of the mutants are temperature sensitive both for growth and for formation of Ψ in the rRNA. In most cases, the impaired growth phenotypes are not relieved by transcription of the rRNA from a polymerase II-driven promoter, indicating the absence of polymerase I-related transcriptional defects. There is little or no abnormal accumulation of pre-rRNAs in these mutants, although preferential inhibition of 18S rRNA synthesis is seen in mutantcbf5D95A, which lacks Ψ in rRNA. A subset of mutations in the Ψ synthase domain impairs association of the altered Cbf5p proteins with selected box H/ACA snoRNAs, suggesting that the functional catalytic domain is essential for that interaction. Our results provide additional evidence that Cbf5p is the Ψ synthase component of box H/ACA snoRNPs and suggest that the pseudouridylation of rRNA, although not absolutely required for cell survival, is essential for the formation of fully functional ribosomes.

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Mesoionic Heterocyclic Compounds as Candidate Messenger RNA Cap Analogue Inhibitors of the Influenza Virus RNA Polymerase Cap-Binding Activity

Antiviral Chemistry and Chemotherapy ◽

10.1177/095632020901900504 ◽

2009 ◽

Vol 19 (5) ◽

pp. 213-218 ◽

Cited By ~ 6

Author(s):

Ian Mickleburgh ◽

Feng Geng ◽

Laurence Tiley

Keyword(s):

Influenza Virus ◽

Rna Polymerase ◽

Messenger Rna ◽

Binding Activity ◽

Endonucleolytic Cleavage ◽

Virus Rna ◽

Fused Ring ◽

Mesoionic Heterocycles ◽

Potential Inhibitors

Background: An unusual feature of influenza viral messenger RNA (mRNA) synthesis is its dependence upon host cell mRNAs as a source of capped RNA primers. A crucial activity of the influenza polymerase is to steal these primers by binding and cleaving the caps from host mRNAs. The recent structural analysis of the cap-binding fragment of the influenza virus PB2 protein has highlighted the importance of the mesoionic properties of the N7-methylguanine (N7mG) component of the mRNA cap in this interaction. Methods: A series of mesoionic heterocycles with 5,6-fused ring systems analogous to the N7mG component of mRNA cap structures were synthesized and examined for the ability to inhibit the cap-binding activity of the influenza virus RNA polymerase complex using a bead-based in vitro cap-binding assay. Results: None of the compounds tested were able to significantly inhibit binding and subsequent endonucleolytic cleavage of a synthetic radiolabelled capped mRNA substrate by recombinant influenza virus polymerase in vitro. Conclusions: Compounds analogous to the mesoionic N7mG component of mRNA cap structures comprise a large class of potential inhibitors of the influenza virus polymerase. Although this preliminary assessment of a small group of related analogues was unsuccessful, further screening of this class of compounds is warranted.

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