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P D Gershon

Publications and source records attributed to P D Gershon.

At least 19 recordsLinked to original sources
PubMed2006

pKa of the mRNA cap-specific 2'-O-methyltransferase catalytic lysine by HSQC NMR detection of a two-carbon probe.

We have characterized the side chain pKa for a single lysine analogue within a 316-residue protein containing 21 lysines and 1678 carbon atoms at natural isotope abundance. To do this, the single reactive cysteine of a K175C mutant of VP39 (the mRNA cap-specific 2'-O-methyltransferase from vaccinia virus) was modified to S-(beta-aminoethyl)cysteine (gamma-thialysine) using freshly prepared (13C)aziridine at room temperature. Modification was accompanied by the rescue of catalytic function at high specific activity. After the fastidious removal of the noncovalently protein-bound aziridine self-polymer using a novel chelating dialysis procedure, signals were monitored by HSQC NMR. Appropriately pH-shifting HSQC NMR peaks were identified in the (13C)aziridine-modified enzyme, corresponding to detection of the two covalently attached (13C)thioethylamino atoms. The identification was strengthened by comparison with the positions and pH shifts of spectral peaks for tripeptide controls, a small molecule aziridine self-polymer mimetic, and a cysteine-minus control enzyme. pH titration of the modified protein indicated an apparent pKa of 8.5, consistent with a perturbed pKa for the catalytic lysine and a model in which the surrounding charged groups direct the lysine epsilon-amino pKa via both local electrostatic environment and orbital directionality.

Amino Acid Sequence↗
PubMed2004

Mechanism of RNA 2'-O-methylation: evidence that the catalytic lysine acts to steer rather than deprotonate the target nucleophile.

The weight of current evidence suggests that RNA 2'-O-MTases employ an S(N)2 mechanism with an in-line attack of the target nucleophile upon the methyl group of the AdoMet cofactor. It has been suggested that, like the phosphohydrolytic enzymes, ribozymes, and nucleic acid polymerases, the RNA 2'-O-MTases initially activate the substrate's attacking hydroxyl oxygen by deprotonation. Here, evidence is presented that the vaccinia virus mRNA cap specific 2'-O-MTase VP39 does not promote RNA 2'-oxyanion formation but that instead it acts by steering a hydroxyl oxygen orbital toward the cofactor methyl center.

Carbon Isotopes↗
PubMed2004

Molecular flexibility and discontinuous translocation of a non-templated polymerase.

Little is known regarding the translocation of non-templated nucleic acid polymerases with respect to single-stranded primers. VP55, the vaccinia virus poly(A) polymerase, translocates as it processively adds a approximately 3-7 adenylate tail to primers possessing only three ribouridylate residues (as an (rU)(2)-N(15)-rU motif), and a approximately 25-30 adenylate tail to primers that are more U-rich. Here, three models were addressed for the translocation of VP55 with respect to its primer, namely: (a) rigid protein/rigid nucleic acid; (b) flexible protein/rigid nucleic acid; (c) rigid protein/flexible nucleic acid. Analysis of free and covalently VP55-attached primers favored either (b) or a version of (c) incorporating a passive steric block, and suggested two regions of relative motion between polymerase and primer. Inclusion of a 6nt uridylate-rich patch at the primer 3' end switched the polymerase from approximately 3-7 nt to approximately 25-30 nt tail addition without affecting initial binding affinity. By synthesizing this patch as a (rU/dC) pool, discontinuous polymerase movements could be detected.

Polynucleotide Adenylyltransferase↗
PubMed2003

Structure of intracellular mature vaccinia virus visualized by in situ atomic force microscopy.

Vaccinia virus, the basis of the smallpox vaccine, is one of the largest viruses to replicate in humans. We have used in situ atomic force microscopy (AFM) to directly visualize fully hydrated, intact intracellular mature vaccinia virus (IMV) virions and chemical and enzymatic treatment products thereof. The latter included virion cores, core-enveloping coats, and core substructures. The isolated coats appeared to be composed of a highly cross-linked protein array. AFM imaging of core substructures indicated association of the linear viral DNA genome with a segmented protein sheath forming an extended approximately 16-nm-diameter filament with helical surface topography; enclosure of this filament within a 30- to 40-nm-diameter tubule which also shows helical topography; and enclosure of the folded, condensed 30- to 40-nm-diameter tubule within the core by a wall covered with peg-like projections. Proteins observed attached to the 30- to 40-nm-diameter tubules may mediate folding and/or compaction of the tubules and/or represent vestiges of the core wall and/or pegs. An accessory "satellite domain" was observed protruding from the intact core. This corresponded in size to isolated 70- to 100-nm-diameter particles that were imaged independently and might represent detached accessory domains. AFM imaging of intact virions indicated that IMV underwent a reversible shrinkage upon dehydration (as much as 2.2- to 2.5-fold in the height dimension), accompanied by topological and topographical changes, including protrusion of the satellite domain. As shown here, the chemical and enzymatic dissection of large, asymmetrical virus particles in combination with in situ AFM provides an informative complement to other structure determination techniques.

Aluminum Silicates↗
PubMed2000

Structural basis of mRNA cap recognition by proteins.

Crystal structures have recently become available for two proteins (VP39 and eIF4E) complexed with their cognate ligand - the mRNA cap. Despite their total structural dissimilarity, both proteins bind N7-methylguanine between two parallel aromatic sidechains. The resulting stacked arrangement governs their high specificity for the alkylated form of the nucleobase.

Alkylation↗
PubMed1999

RNA binding characteristics and overall topology of the vaccinia poly(A) polymerase-processivity factor-primer complex.

The vaccinia virus-encoded heterodimer responsible for poly(A) tail elongation comprises a polyadenylylation catalytic subunit (VP55) and associated processivity factor (VP39). We show that monomeric VP39's affinity for RNA homopolymers follows the hierarchy poly(I) >poly(U) >>poly(G) >poly(A) >poly(C), that the heterodimer interacts stably with 40-45 nucleotide nucleic acid segments, and that its homopolymer preference for polyadenylylation priming is comparable to the VP39 affinity hierarchy (above). For oligonucleotide ligands possessing the previously-identified (rU)2-(N)25-rU heterodimer-binding motif, the heterodimer's affinity and base-type preference are mediated via both the (rU)2and rU portions, with the greater contribution coming from (rU)2. VP39's R107 sidechain contributes to specificity at the downstream rU. Substitution of each ribouridylate of the motif with either ribothymidine or 4-thiodeoxythymidine indicated that the downstream rU interacts with both heterodimer subunits, whereas the upstream (rU)2interacts only with VP55. A 'crosslinking SELEX' approach indicated VP39-base proximity around position -10 of a 4-thioribouridine/deoxycytidine ligand pool. Upon incubating the heterodimer with a panel of identical-sequence oligonucleotides derivatized with azidophenacyl bromide at various phosphate positions, those derivatized at positions -11 to -21 photocrosslinked to both subunits in a coordinated manner. This region may therefore pass through a 'cleft' or enclosed 'channel' at the subunit interface.

Binding Sites↗
PubMed1999

mRNA cap recognition: dominant role of enhanced stacking interactions between methylated bases and protein aromatic side chains.

We have determined, by high resolution x-ray analysis, 10 structures comprising the mRNA cap-specific methyltransferase VP39 or specific mutants thereof in the presence of methylated nucleobase analogs (N1-methyladenine, N3-methyladenine, N1-methylcytosine, N3-methylcytosine) and their unmethylated counterparts, or nucleoside N7-methylguanosine. Together with solution affinity studies and previous crystallographic data for N7-methylguanosine and its phosphorylated derivatives, these data demonstrate that only methylated, positively charged bases are bound, indicating that their enhanced stacking with two aromatic side chains of VP39 (Tyr 22 and Phe 180) plays a dominant role in cap recognition. Four key features characterize this stacking interaction: (i) near perfect parallel alignment between the sandwiched methylated bases and aromatic side chains, (ii) substantial areas of overlap in the two-stacked rings, (iii) a 3.4-A interplanar spacing within the overlapping region, and (iv) positive charge in the heterocyclic nucleobase.

Binding Sites↗
PubMed1999

A polyadenylylation-specific RNA-contact site on the surface of the bifunctional vaccinia virus RNA modifying protein VP39 that is distinct from the mRNA 5' end-binding "cleft".

VP39 is a bifunctional mRNA-modifying protein that acts as both an mRNA cap-specific 2'-O-methyltransferase and a processivity factor for VP55, the vaccinia poly(A) polymerase catalytic subunit. Although regions of the protein surface required for methyltransferase function are well defined, it has been unclear whether the protein polyadenylylation function requires direct RNA contact and, if so, where the contact site(s) might be located on the protein surface. Here, we show that the VP55-VP39 heterodimer forms a stable complex with a 50mer oligonucleotide bearing a U2-N25-U motif, as opposed to the U2-N15-U motif that is optimal for stable complex formation with VP55 alone. An oligonucleotide bearing a U2-N25-U motif in which the downstream U residue is replaced with 4thioU can be efficiently photocrosslinked to VP39, but only in the context of the VP55-VP39 heterodimer. By partial proteolysis of end-labeled VP39, the site of oligonucleotide photocrosslinking was localized to the region of VP39 between residues Lys90 and Arg122. Peptide microsequencing and confirmatory mutagenesis identified the side-chain of Arg107 as the photocrosslinking site. Substitution of this residue with lysine abolished photocrosslinking entirely, consistent with the established RNA binding role of arginine in other RNA-binding proteins. This study provides clear evidence for a polyadenylylation-specific RNA-contact site on the surface of VP39, which is distinct from the RNA-binding methyltransferase "cleft" characterized in recent crystallographic and biochemical studies.

Base Sequence↗
PubMed1998

Evidence that the RNA methylation and poly(A) polymerase stimulatory activities of vaccinia virus protein VP39 do not impinge upon one another.

Vaccinia protein VP39 has two RNA modifying activities. In monomeric form, it acts as an mRNA cap-specific 2'-O-methyltransferase, specifically modifying the ribose moiety of the first transcribed nucleotide of m7G-capped mRNA. In association with VP55, the catalytic subunit of the vaccinia poly(A) polymerase, VP39 facilitates the rapid elongation of poly(A) tails that are already greater than approximately 35 nt in length. Introducing new assays, we provide evidence that substrates for each of VP39's two activities do not detectably modulate the converse reaction and that VP39's 2'-O-methyltransferase activity is not significantly affected by its association with VP55. In an electrophoretic mobility shift assay, VP39 interacted with a short (5 nucleotide) RNA only when the latter was m7G-capped. Complexes with longer (22 nucleotide) RNAs were more stable (i.e., cap-independent) but were further stabilized by the presence of an m7G cap. An additional complex was observed at elevated RNA:protein molar ratios, indicating the presence of two RNA binding sites per VP39 molecule. Interaction at one of these sites was stabilized by the cap structure. Additional experiments indicated that RNA molecules undergoing poly(A) tail elongation by the VP55-VP39 heterodimer are not favored as cap-methylation substrates.

Binding Sites↗
PubMed1998

Cap ribose methylation of c-mos mRNA stimulates translation and oocyte maturation in Xenopus laevis.

In Xenopus oocytes, progesterone stimulates the cytoplasmic polyadenylation and resulting translational activation of c-mos mRNA, which is necessary for the induction of oocyte maturation. Although details of the biochemistry of polyadenylation are beginning to emerge, the mechanism by which 3' poly(A) addition stimulates translation initiation is enigmatic. A previous report showed that polyadenylation induced cap-specific 2'-O-methylation, and suggested that this 5' end modification was important for translational activation. Here, we demonstrate that injected c-mos RNA undergoes polyadenylation and cap ribose methylation. Inhibition of this methylation by S-isobutylthioadenosine (SIBA), a methyltransferase inhibitor, has little effect on progesterone-induced c-mos mRNA polyadenylation or general protein synthesis, but prevents the synthesis of Mos protein as well as oocyte maturation. Maturation can be rescued, however, by the injection of factors that act downstream of Mos, such as cyclin A and B mRNAs. Most importantly, we show that the translational efficiency of injected mRNAs containing cap-specific 2'-O-methylation (cap I) is significantly enhanced compared to RNAs that do not contain the methylated ribose (cap 0). These results suggest that cap ribose methylation of c-mos mRNA is important for translational recruitment and for the progression of oocytes through meiosis.

Animals↗
PubMed1998

Recognition of capped RNA substrates by VP39, the vaccinia virus-encoded mRNA cap-specific 2'-O-methyltransferase.

We have investigated the interaction of VP39, the vaccinia-encoded mRNA cap-specific 2'-O-methyltransferase, with its capped RNA substrate. Two sites on the protein surface, responsible for binding the terminal cap nucleotide (m7G) and cap-proximal RNA, were characterized, and a third (downstream RNA binding) site was identified. Regarding the crystallographically defined m7G binding pocket, VP39 showed significant activity with adenine-capped RNA. Although VP39 mutants lacking specific m7G-contact side chains within the pocket showed reduced catalytic activity, none was transformed into a cap-independent RNA methyltransferase. Moreover, each retained a preference for m7G and A over unmethylated G as the terminal cap nucleotide, indicating a redundancy of m7G-contact residues able to confer cap-type specificity. Despite containing the 2'-O-methylation site, m7GpppG (cap dinucleotide) could not be methylated by VP39, but m7GpppGUbiotinp could. This indicated the minimum-length 2'-O-methyltransferase substrate to be either m7GpppGp, m7GpppGpN, or m7GpppGpNp. RNA-protein contacts immediately downstream of the m7GpppG moiety were found to be pH-sensitive. This was detectable only in the context of a weakened interaction of near-minimum-length substrates with VP39's m7G binding pocket (through the use of either adenine-capped substrate or a VP39 pocket mutant), as a dramatic elevation of KM at pH values above 7.5. KM values for substrates with RNA chain lengths of 2-6 nt were between 160 and 230 nM, but dropped to 9-15 nM upon increasing chain lengths to 20-50 nt. This suggested the binding of regions of the RNA substrate >6 nt from the 5' terminus to a previously unknown site on the VP39 surface.

Amino Acid Substitution↗
PubMed1998

Structural basis for sequence-nonspecific recognition of 5'-capped mRNA by a cap-modifying enzyme.

Sequence-nonspecific binding of RNA, recognition of a 7-methylguanosine 5' mRNA cap, and methylation of a nucleic acid backbone are three crucial and ubiquitous events in eukaryotic nucleic acid processing and function. These three events occur concurrently in the modification of vaccinia transcripts by the methyltransferase VP39. We report the crystal structure of a ternary complex comprising VP39, coenzyme product S-adenosylhomocysteine, and a 5' m7 G-capped, single-stranded RNA hexamer. This structure reveals a novel and general mechanism for sequence-non-specific recognition of the mRNA transcript in which the protein interacts solely with the sugar-phosphate backbone of a short, single-stranded RNA helix. This report represents the first direct and detailed view of a protein complexed with single-stranded RNA or 5'-capped mRNA.

Binding Sites↗
PubMed1997

Specific recognition of an rU2-N15-rU motif by VP55, the vaccinia virus poly(A) polymerase catalytic subunit.

VP55, the vaccinia poly(A) polymerase catalytic subunit, interacts with oligonucleotide primers via two uridylate recognition sites (Deng, L., and Gershon, P. D. (1997) EMBO J. 16, 1103-1113). Here, we show that the cognate RNA sequence comprises a 5'-rU2-N15-rU-3' motif (where N = any deoxyribo or ribonucleotide), embedded within oligonucleotide primers 29-30 nucleotides (nt), or greater, in length. Nine residues separate the 3'-most ribouridylate of the optimally positioned motif from the primer 3'-OH. A ribose sugar at the extreme 3'-terminal nucleotide of the primer is absolutely required for VP55's adenylyltransferase activity, but not for stable VP55-RNA interaction. A ribose at position -3 markedly stimulates both adenylyltransferase activity and stable binding. The use of uridine analogs indicated (i) those functional groups of the uracil base which contribute to stable VP55-primer interaction, and (ii) that VP55's ability to discriminate uracil from cytosine stems largely from the requirement for a protonated N3 nitrogen within the pyrimidine ring. The rU2-N15-rU motif was identified within the uridylate-rich 3' end of a naturally occurring vaccinia mRNA. However, oligonucleotides whose only internal uridylates comprised the motif supported only a 3-5-nt processive burst of oligo(A) tail addition, as opposed to the approximately 30-35-nt burst observed with the naturally occurring 3' end.

Binding Sites↗
PubMed1997

The surface region of the bifunctional vaccinia RNA modifying protein VP39 that interfaces with Poly(A) polymerase is remote from the RNA binding cleft used for its mRNA 5' cap methylation function.

VP39 is a single-domain, bifunctional viral protein, which acts at both ends of nascent mRNA. At the 5' end, it acts as a cap-specific 2'-O-methyltransferase. At the 3' end, it acts as a poly(A) polymerase processivity factor, requiring its direct association with poly(A) polymerase. Although crystallographic and biochemical data show the catalytic center and associated binding sites for VP39's methyltransferase function to be juxtaposed around a superficial cleft on the protein surface, surface regions required for VP39's mRNA 3' end modifying functions are not known. Here, we identify a surface region that interfaces directly with poly(A) polymerase, taking three independent approaches: (i) development of a direct in vitro dimerization assay, which is applied to numerous VP39 point mutants; (ii) identification of sites within VP39 that become protected from protease cleavage upon dimerization and further mutagenesis based upon these data; (iii) site-specific photo-cross-linking of VP39 to VP55. We find that the dimerization interface lies on a surface region remote from the methyltransferase cleft and contains a 3-5-residue "hot-spot," which is very sensitive to amino acid substitutions. Various other sites within VP39 consistently became hypersensitive to protease cleavage upon interaction with VP55, indicating the occurrence of extensive conformational changes.

Amino Acid Sequence↗
PubMed1997

Interplay of two uridylate-specific RNA binding sites in the translocation of poly(A) polymerase from vaccinia virus.

The VP55 (catalytic) subunit of vaccinia virus heterodimeric poly(A) polymerase (PAP) contacts 31-40 nucleotide segments of RNA in a uridylate-dependent manner, and effects the rapid, processive addition of a 30 nt oligo(A) tail. Here, the minimum size of uridylate-containing RNA required for stable VP55 interaction was refined to 33-34 nt. VP55 binding experiments using a set of sixteen 34 nt DNA-RNA chimeras, each containing a differently positioned tetra-uridylate cluster within an oligo(dC) background, indicated that the protein contacts uridylates at two positions within the oligonucleotide. Combination of two optimally positioned tetra-uridylate clusters into a single oligonucleotide fully restored the properties of an optimal substrate, rU34, in VP55 binding and salt-resistant polyadenylylation. The positions of the two uridylate interaction sites, approximately 10 and approximately 25 nt from the oligonucleotide 3' OH, were confirmed using a selection scheme employing dC-rU oligonucleotide chimera pools. These and additional data suggest a mechanism for polymerase translocation with respect to RNA comparable with inchworming models of transcriptional elongation. In selection experiments incorporating the PAP-associated processivity factor VP39, the latter was shown to replace the 3' OH-distal uridylate contact site with one approximately 10 nt further upstream.

Binding Sites↗
PubMed1996

The 1.85 A structure of vaccinia protein VP39: a bifunctional enzyme that participates in the modification of both mRNA ends.

VP39 is a bifunctional vaccinia virus protein that acts as both an mRNA cap-specific RNA 2'-O-methyltransferase and a poly(A) polymerase processivity factor. Here, we report the 1.85 A crystal structure of a VP39 variant complexed with its AdoMet cofactor. VP39 comprises a single core domain with structural similarity to the catalytic domains of other methyltransferases. Surface features and mutagenesis data suggest two possible RNA-binding sites with novel underlying architecture, one of which forms a cleft spanning the region adjacent to the methyltransferase active site. This report provides a prototypic structure for an RNA methyltransferase, a protein that interacts with the mRNA 5' cap, and an intact poxvirus protein.

Binding Sites↗