scholarly journals Transcriptional Control and mRNA Capping by the GDP Polyribonucleotidyltransferase Domain of the Rabies Virus Large Protein

Viruses ◽  
2019 ◽  
Vol 11 (6) ◽  
pp. 504 ◽  
Author(s):  
Tomoaki Ogino ◽  
Todd J. Green
Keyword(s):  
Rabies Virus ◽  
Transcription Initiation ◽  
Transcriptional Control ◽  
Rna Synthesis ◽  
Molecular Mechanisms ◽  
Antiviral Agents ◽  
L Protein ◽  
Mrna Capping ◽  
Capping Enzyme

Rabies virus (RABV) is a causative agent of a fatal neurological disease in humans and animals. The large (L) protein of RABV is a multifunctional RNA-dependent RNA polymerase, which is one of the most attractive targets for developing antiviral agents. A remarkable homology of the RABV L protein to a counterpart in vesicular stomatitis virus, a well-characterized rhabdovirus, suggests that it catalyzes mRNA processing reactions, such as 5′-capping, cap methylation, and 3′-polyadenylation, in addition to RNA synthesis. Recent breakthroughs in developing in vitro RNA synthesis and capping systems with a recombinant form of the RABV L protein have led to significant progress in our understanding of the molecular mechanisms of RABV RNA biogenesis. This review summarizes functions of RABV replication proteins in transcription and replication, and highlights new insights into roles of an unconventional mRNA capping enzyme, namely GDP polyribonucleotidyltransferase, domain of the RABV L protein in mRNA capping and transcription initiation.

scholarly journals Formation of Guanosine(5′)Tetraphospho(5′)Adenosine Cap Structure by an Unconventional mRNA Capping Enzyme of Vesicular Stomatitis Virus

2008 ◽  
Vol 82 (15) ◽  
pp. 7729-7734 ◽  
Author(s):  
Tomoaki Ogino ◽  
Amiya K. Banerjee
Keyword(s):  
Vesicular Stomatitis Virus ◽  
In Vitro Transcription ◽  
Viral Mrna ◽  
Viral Mrnas ◽  
L Protein ◽  
Transcription System ◽  
Mrna Capping ◽  
Capping Enzyme ◽  
Vesicular Stomatitis

ABSTRACT The RNA-dependent RNA polymerase L protein of vesicular stomatitis virus (VSV) elicits GTPase and RNA:GDP polyribonucleotidyltransferase (PRNTase) activities to produce a 5′-cap core structure, guanosine(5′)triphospho(5′)adenosine (GpppA), on viral mRNAs. Here, we report that the L protein produces an unusual cap structure, guanosine(5′)tetraphospho(5′)adenosine (GppppA), that is formed by the transfer of the 5′-monophosphorylated viral mRNA start sequence to GTP by the PRNTase activity before the removal of the γ-phosphate from GTP by GTPase. Interestingly, GppppA-capped and polyadenylated full-length mRNAs were also found to be synthesized by an in vitro transcription system with the native VSV RNP.

scholarly journals 5′-Phospho-RNA Acceptor Specificity of GDP Polyribonucleotidyltransferase of Vesicular Stomatitis Virus in mRNA Capping

2017 ◽  
Vol 91 (6) ◽  
Author(s):  
Minako Ogino ◽  
Tomoaki Ogino
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Rna Viruses ◽  
Antiviral Agents ◽  
Host Cells ◽  
Hydroxyl Group ◽  
L Protein ◽  
Acceptor Specificity ◽  
Mrna Capping ◽  
Capping Enzyme ◽  
Vesicular Stomatitis

ABSTRACT The GDP polyribonucleotidyltransferase (PRNTase) domain of the multifunctional L protein of rhabdoviruses, such as vesicular stomatitis virus (VSV) and rabies virus, catalyzes the transfer of 5′-phospho-RNA (pRNA) from 5′-triphospho-RNA (pppRNA) to GDP via a covalent enzyme-pRNA intermediate to generate a 5′-cap structure (GpppA). Here, using an improved oligo-RNA capping assay with the VSV L protein, we showed that the Michaelis constants for GDP and pppAACAG (VSV mRNA-start sequence) are 0.03 and 0.4 μM, respectively. A competition assay between GDP and GDP analogues in the GpppA formation and pRNA transfer assay using GDP analogues as pRNA acceptors indicated that the PRNTase domain recognizes the C-2-amino group, but not the C-6-oxo group, N-1-hydrogen, or N-7-nitrogen, of GDP for the cap formation. 2,6-Diaminopurine-riboside (DAP), 7-deazaguanosine (7-deaza-G), and 7-methylguanosine (m7G) diphosphates efficiently accepted pRNA, resulting in the formation of DAPpppA, 7-deaza-GpppA, and m7GpppA (cap 0), respectively. Furthermore, either the 2′- or 3′-hydroxyl group of GDP was found to be required for efficient pRNA transfer. A 5′-diphosphate form of antiviral ribavirin weakly inhibited the GpppA formation but did not act as a pRNA acceptor. These results indicate that the PRNTase domain has a unique guanosine-binding mode different from that of eukaryotic mRNA capping enzyme, guanylyltransferase. IMPORTANCE mRNAs of nonsegmented negative-strand (NNS) RNA viruses, such as VSV, possess a fully methylated cap structure, which is required for mRNA stability, efficient translation, and evasion of antiviral innate immunity in host cells. GDP polyribonucleotidyltransferase (PRNTase) is an unconventional mRNA capping enzyme of NNS RNA viruses that is distinct from the eukaryotic mRNA capping enzyme, guanylyltransferase. In this study, we studied the pRNA acceptor specificity of VSV PRNTase using various GDP analogues and identified chemical groups of GDP as essential for the substrate activity. The findings presented here are useful not only for understanding the mechanism of the substrate recognition with PRNTase but also for designing antiviral agents targeting this enzyme.

scholarly journals An In Vitro RNA Synthesis Assay for Rabies Virus Defines Ribonucleoprotein Interactions Critical for Polymerase Activity

2016 ◽  
Vol 91 (1) ◽  
Author(s):  
Benjamin Morin ◽  
Bo Liang ◽  
Erica Gardner ◽  
Robin A. Ross ◽  
Sean P. J. Whelan
Keyword(s):  
Rabies Virus ◽  
Nucleocapsid Protein ◽  
Rna Synthesis ◽  
Recombinant Baculovirus ◽  
Promoter Sequence ◽  
Size Exclusion ◽  
Rdrp Activity ◽  
Content Type ◽  
L Protein

ABSTRACT We report an in vitro RNA synthesis assay for the RNA-dependent RNA polymerase (RdRP) of rabies virus (RABV). We expressed RABV large polymerase protein (L) in insect cells from a recombinant baculovirus vector and the phosphoprotein cofactor (P) in Escherichia coli and purified the resulting proteins by affinity and size exclusion chromatography. Using chemically synthesized short RNA corresponding to the first 19 nucleotides (nt) of the rabies virus genome, we demonstrate that L alone initiates synthesis on naked RNA and that P serves to enhance the initiation and processivity of the RdRP. The L-P complex lacks full processivity, which we interpret to reflect the lack of the viral nucleocapsid protein (N) on the template. Using this assay, we define the requirements in P for stimulation of RdRP activity as residues 11 to 50 of P and formally demonstrate that ribavirin triphosphate (RTP) inhibits the RdRP. By comparing the properties of RABV RdRP with those of the related rhabdovirus, vesicular stomatitis virus (VSV), we demonstrate that both polymerases can copy the heterologous promoter sequence. The requirements for engagement of the N-RNA template of VSV by its polymerase are provided by the C-terminal domain (CTD) of P. A chimeric RABV P protein in which the oligomerization domain (OD) and the CTD were replaced by those of VSV P stimulated RABV RdRP activity on naked RNA but was insufficient to permit initiation on the VSV N-RNA template. This result implies that interactions between L and the template N are also required for initiation of RNA synthesis, extending our knowledge of ribonucleoprotein interactions that are critical for gene expression. IMPORTANCE The current understanding of the structural and functional significance of the components of the rabies virus replication machinery is incomplete. Although structures are available for the nucleocapsid protein in complex with RNA, and also for portions of P, information on both the structure and function of the L protein is lacking. This study reports the expression and purification of the full-length L protein of RABV and the characterization of its RdRP activity in vitro. The study provides a new assay that has utility for screening inhibitors and understanding their mechanisms of action, as well as defining new interactions that are required for RdRP activity.

High resolution mapping of nucleic acid containing complexes in vitro and in situ

1996 ◽  
Vol 54 ◽  
pp. 48-49
Author(s):  
D. P. Bazett-Jones ◽  
M. J. Hendzel
Keyword(s):  
Nucleic Acid ◽  
High Resolution ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Heavy Atom ◽  
Regulation Of Transcription ◽  
High Exposure ◽  
Resolution Mapping ◽  
Tata Sequence

Structural analysis of combinations of nucleosomes and transcription factors on promoter and enhancer elements is necessary in order to understand the molecular mechanisms responsible for the regulation of transcription initiation. Such complexes are often not amenable to study by high resolution crystallographic techniques. We have been applying electron spectroscopic imaging (ESI) to specific problems in molecular biology related to transcription regulation. There are several advantages that this technique offers in studies of nucleoprotein complexes. First, an intermediate level of spatial resolution can be achieved because heavy atom contrast agents are not necessary. Second, mass and stoichiometric relationships of protein and nucleic acid can be estimated by phosphorus detection, an element in much higher proportions in nucleic acid than protein. Third, wrapping or bending of the DNA by the protein constituents can be observed by phosphorus mapping of the complexes. Even when ESI is used with high exposure of electrons to the specimen, important macromolecular information may be provided. For example, an image of the TATA binding protein (TBP) bound to DNA is shown in the Figure (top panel). It can be seen that the protein distorts the DNA away from itself and much of its mass sits off the DNA helix axis. Moreover, phosphorus and mass estimates demonstrate whether one or two TBP molecules interact with this particular promoter TATA sequence.

Analysis of Saccharomyces cerevisiae his3 transcription in vitro: biochemical support for multiple mechanisms of transcription

1990 ◽  
Vol 10 (6) ◽  
pp. 2832-2839
Author(s):  
A S Ponticelli ◽  
K Struhl
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Activation ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Initiation Site ◽  
Activator Proteins ◽  
Nuclear Extracts ◽  
Transcription In Vitro

The promoter region of the Saccharomyces cerevisiae his3 gene contains two TATA elements, TC and TR, that direct transcription initiation to two sites designated +1 and +13. On the basis of differences between their nucleotide sequences and their responsiveness to upstream promoter elements, it has previously been proposed that TC and TR promote transcription by different molecular mechanisms. To begin a study of his3 transcription in vitro, we used S. cerevisiae nuclear extracts together with various DNA templates and transcriptional activator proteins that have been characterized in vivo. We demonstrated accurate transcription initiation in vitro at the sites used in vivo, transcriptional activation by GCN4, and activation by a GAL4 derivative on various gal-his3 hybrid promoters. In all cases, transcription stimulation was dependent on the presence of an acidic activation region in the activator protein. In addition, analysis of promoters containing a variety of TR derivatives indicated that the level of transcription in vitro was directly related to the level achieved in vivo. The results demonstrated that the in vitro system accurately reproduced all known aspects of in vivo his3 transcription that depend on the TR element. However, in striking contrast to his3 transcription in vivo, transcription in vitro yielded approximately 20 times more of the +13 transcript than the +1 transcript. This result was not due to inability of the +1 initiation site to be efficiently utilized in vitro, but rather it reflects the lack of TC function in vitro. The results support the idea that TC and TR mediate transcription from the wild-type promoter by distinct mechanisms.

scholarly journals Molecular Mechanisms Underlying the Positive Stringent Response of the Bacillus subtilis ilv-leu Operon, Involved in the Biosynthesis of Branched-Chain Amino Acids

2008 ◽  
Vol 190 (18) ◽  
pp. 6134-6147 ◽  
Author(s):  
Shigeo Tojo ◽  
Takenori Satomura ◽  
Kanako Kumamoto ◽  
Kazutake Hirooka ◽  
Yasutaro Fujita
Keyword(s):  
Amino Acids ◽  
Bacillus Subtilis ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Stringent Response ◽  
Branched Chain Amino Acids ◽  
Base Substitution ◽  
Branched Chain

ABSTRACT Branched-chain amino acids are the most abundant amino acids in proteins. The Bacillus subtilis ilv-leu operon is involved in the biosynthesis of branched-chain amino acids. This operon exhibits a RelA-dependent positive stringent response to amino acid starvation. We investigated this positive stringent response upon lysine starvation as well as decoyinine treatment. Deletion analysis involving various lacZ fusions revealed two molecular mechanisms underlying the positive stringent response of ilv-leu, i.e., CodY-dependent and -independent mechanisms. The former is most likely triggered by the decrease in the in vivo concentration of GTP upon lysine starvation, GTP being a corepressor of the CodY protein. So, the GTP decrease derepressed ilv-leu expression through detachment of the CodY protein from its cis elements upstream of the ilv-leu promoter. By means of base substitution and in vitro transcription analyses, the latter (CodY-independent) mechanism was found to comprise the modulation of the transcription initiation frequency, which likely depends on fluctuation of the in vivo RNA polymerase substrate concentrations after stringent treatment, and to involve at least the base species of adenine at the 5′ end of the ilv-leu transcript. As discussed, this mechanism is presumably distinct from that for B. subtilis rrn operons, which involves changes in the in vivo concentration of the initiating GTP.

scholarly journals Sir2 Silences Gene Transcription by Targeting the Transition between RNA Polymerase II Initiation and Elongation

2008 ◽  
Vol 28 (12) ◽  
pp. 3979-3994 ◽  
Author(s):  
Lu Gao ◽  
David S. Gross
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Transcriptional Silencing ◽  
Pol Ii ◽  
Initiation Factors ◽  
Lysine Deacetylase ◽  
Evolutionarily Conserved ◽  
Mrna Capping ◽  
Capping Enzyme

ABSTRACT It is well accepted that for transcriptional silencing in budding yeast, the evolutionarily conserved lysine deacetylase Sir2, in concert with its partner proteins Sir3 and Sir4, establishes a chromatin structure that prevents RNA polymerase II (Pol II) transcription. However, the mechanism of repression remains controversial. Here, we show that the recruitment of Pol II, as well as that of the general initiation factors TBP and TFIIH, occurs unimpeded to the silent HMR a 1 and HMLα1/HMLα2 mating promoters. This, together with the fact that Pol II is Ser5 phosphorylated, implies that SIR-mediated silencing is permissive to both preinitiation complex (PIC) assembly and transcription initiation. In contrast, the occupancy of factors critical to both mRNA capping and Pol II elongation, including Cet1, Abd1, Spt5, Paf1C, and TFIIS, is virtually abolished. In agreement with this, efficiency of silencing correlates not with a restriction in Pol II promoter occupancy but with a restriction in capping enzyme recruitment. These observations pinpoint the transition between polymerase initiation and elongation as the step targeted by Sir2 and indicate that transcriptional silencing is achieved through the differential accessibility of initiation and capping/elongation factors to chromatin. We compare Sir2-mediated transcriptional silencing to a second repression mechanism, mediated by Tup1. In contrast to Sir2, Tup1 prevents TBP, Pol II, and TFIIH recruitment to the HMLα1 promoter, thereby abrogating PIC formation.

scholarly journals Pausing controls branching between productive and non-productive pathways during initial transcription

2017 ◽  
Author(s):  
David Dulin ◽  
David L. V. Bauer ◽  
Anssi M. Malinen ◽  
Jacob J. W. Bakermans ◽  
Martin Kaller ◽  
...  
Keyword(s):  
Single Molecule ◽  
Transcription Initiation ◽  
Rna Synthesis ◽  
Molecular Mechanisms ◽  
Dependent Manner ◽  
Single Molecule Fret ◽  
Bacterial Rna ◽  
Tightly Coupled ◽  
Relationship Of ◽  
The Relationship

AbstractTranscription in bacteria is controlled by multiple molecular mechanisms that precisely regulate gene expression. Recently, initial RNA synthesis by the bacterial RNA polymerase (RNAP) has been shown to be interrupted by pauses; however, the pausing determinants and the relationship of pausing with productive and abortive RNA synthesis remain poorly understood. Here, we employed single-molecule FRET and biochemical analysis to disentangle the pausing-related pathways of bacterial initial transcription. We present further evidence that region σ3.2 constitutes a barrier after the initial transcribing complex synthesizes a 6-nt RNA (ITC6), halting transcription. We also show that the paused ITC6 state acts as a checkpoint that directs RNAP, in an NTP-dependent manner, to one of three competing pathways: productive transcription, abortive RNA release, or a new unscrunching/scrunching pathway that blocks transcription initiation. Our results show that abortive RNA release and DNA unscrunching are not as tightly coupled as previously thought.

SIRT1 regulates sphingolipid metabolism and neural differentiation of mouse embryonic stem cells through c-Myc- SMPDL3B

2021 ◽  
Author(s):  
Wei Fan ◽  
Shuang Tang ◽  
Xiaojuan Fan ◽  
Yi Fang ◽  
Xiaojiang Xu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Neural Differentiation ◽  
Transcriptional Control ◽  
Molecular Mechanisms ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
Sphingolipid Metabolism

AbstractSphingolipids are important structural components of cell membranes and prominent signaling molecules controlling cell growth, differentiation, and apoptosis. Sphingolipids are particularly abundant in the brain, and defects in sphingolipid degradation are associated with several human neurodegenerative diseases. However, molecular mechanisms governing sphingolipid metabolism remain unclear. Here we report that sphingolipid degradation is under transcriptional control of SIRT1, a highly conserved mammalian NAD+-dependent protein deacetylase, in mouse embryonic stem cells (mESCs). Deletion of SIRT1 results in accumulation of sphingomyelin in mESCs, primarily due to reduction of SMPDL3B, a GPI-anchored plasma membrane bound sphingomyelin phosphodiesterase. Mechanistically, SIRT1 regulates transcription of Smpdl3b through c-Myc. Functionally, SIRT1 deficiency-induced accumulation of sphingomyelin increases membrane fluidity and impairs neural differentiation in vitro and in vivo. Our findings discover a key regulatory mechanism for sphingolipid homeostasis and neural differentiation, further imply that pharmacological manipulation of SIRT1-mediated sphingomyelin degradation might be beneficial for treatment of human neurological diseases.

A nuclease-hypersensitive element of the human c-myc promoter interacts with a transcription initiation factor

1989 ◽  
Vol 9 (11) ◽  
pp. 5123-5133
Author(s):  
E H Postel ◽  
S E Mango ◽  
S J Flint
Keyword(s):  
Transcription Factor ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Initiation Factor ◽  
Mobility Shift ◽  
Cis Acting ◽  
Factor Binding ◽  
Myc Oncogene ◽  
Transcription In Vitro

Transcription of the human c-myc oncogene is elaborately regulated, but the relevant molecular mechanisms are not yet understood. To begin to define elements and enzyme systems responsible for c-myc transcription in vitro, we partially purified a transcription factor essential for efficient and accurate in vitro initiation from the principal myc promoter, P2. DNA mobility shift assays located the factor binding domain at -142 to -115 with respect to the P1 promoter. This region contains pur/pyr sequences (predominantly purines in one strand), nuclease-hypersensitive sites (U. Siebenlist, L. Henninghausen, J. Battey, and P. Leder, Cell 37:381-391, 1984; C. Boles and M. Hogan, Biochemistry 26:367-376, 1987), and a triple-helix-forming element (M. Cooney, G. Czernuszewicz, E. Postel, S. Flint, and M. Hogan, Science 241:456-459, 1988). Methylation interference mapping established that the factor, termed PuF, directly contacts the repeated palindromic sequence GGGTGGG of the -142/-115 element. The interaction of PuF with this cis-acting element is necessary for P2 transcription in vitro, for (i) deletion of this 5' region from the myc promoter greatly reduced transcription efficiency and (ii) a synthetic duplex oligonucleotide corresponding to the -142/-115 sequence completely repressed c-myc transcription in the presence of the partially purified factor. These observations lend support to the hypothesis that pur/pyr sequences perform important biological roles in the regulation of c-myc gene expression, most likely by serving as transcription factor binding sites.

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