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Paul D Gershon

Publications and source records attributed to Paul D Gershon.

7 recordsLinked to original sources
PubMed2006

Crystal structures of the vaccinia virus polyadenylate polymerase heterodimer: insights into ATP selectivity and processivity.

Polyadenylation of mRNAs in poxviruses, crucial for virion maturation, is carried out by a poly(A) polymerase heterodimer composed of a catalytic component, VP55, and a processivity factor, VP39. The ATP-gamma-S bound and unbound crystal structures of the vaccinia polymerase reveal an unusual architecture for VP55 that comprises of N-terminal, central or catalytic, and C-terminal domains with different topologies and that differs from many polymerases, including the eukaryotic poly(A) polymerases. Residues in the active site of VP55, located between the catalytic and C-terminal domains, make specific interactions with the adenine of the ATP analog, establishing the molecular basis of ATP recognition. VP55's concave surface docks the globular VP39. A model for RNA primer binding that involves all three VP55 domains and VP39 is proposed. The model supports biochemical evidence that VP39 functions as a processivity factor by partially enclosing the RNA primer at the heterodimer interface.

Adenosine Triphosphate↗
PubMed2004

Studying vaccinia virus RNA processing in vitro.

The study and use of vaccinia virus-derived RNA-modifying proteins has made a significant contribution to molecular biology. Here, the purification and assay of two such proteins, comprising the vaccinia poly(A) polymerase/cap-specific mRNA 2'-O-methyltransferase, is described.

Cell Line↗
PubMed2002

The positive transcription elongation factor activity of the vaccinia virus J3 protein is independent from its (nucleoside-2'-O-) methyltransferase and poly(A) polymerase stimulatory functions.

Previous genetic and biochemical experiments have shown that the vaccinia virus J3 protein has three different roles in mRNA synthesis and modification. First, J3 is a (nucleoside-2'-O-)methyltransferase which methylates the 2' position of the first transcribed nucleotide, thus converting a cap-0 to a cap-1 structure at the 5' ends of mRNAs. Second, J3 is a processivity factor for the virus coded poly(A) polymerase. Third, J3 has recently been shown to have intermediate and late gene positive transcription elongation factor activity in vivo. Previous experiments have shown that the poly(A) polymerase stimulatory activity and the (nucleoside-2'-O-)methyltransferase activity are two independent functions of the protein that can be genetically separated through site-directed mutagenesis. In this article, the relationship between the J3-mediated transcription elongation activity and the two other functions of the protein was investigated by constructing several site-directed mutant viruses that contain specific defects in either methyltransferase or poly(A) polymerase processivity functions. The results demonstrate that the J3 positive transcription elongation factor activity is a third independent function of the protein that is genetically separable from its two other functions in mRNA modification. The results also show that neither the poly(A) polymerase stimulatory nor the methyltransferase activities of the J3 protein is essential for virus growth in cell culture.

Methyltransferases↗
PubMed2002

The "cap-binding slot" of an mRNA cap-binding protein: quantitative effects of aromatic side chain choice in the double-stacking sandwich with cap.

The N7-methylguanine portion of the mRNA cap structure interacts with cap-binding proteins via an unusual double-stacking arrangement in which the positively charged cap is sandwiched between two parallel-oriented aromatic protein side chains. Three-dimensional costructures of cap with two mRNA cap-binding proteins, namely, translational initiation factor eIF4E and VP39 (the vaccinia virus-encoded mRNA cap-specific 2'-O-methyltransferase), have heretofore been reported. Despite striking similarities between the two proteins in the double stack with the cap, the stack differs most notably in the species of stacked side chain donated by the protein. Whereas eIF4E employs two tryptophans, VP39 uses a tyrosine and a phenylalanine. Here, we have generated tryptophan substitutions in VP39. Tryptophan substitution was shown, crystallographically, not to disrupt the maintenance of a bona fide parallel stack. However, the single-tryptophan and double-tryptophan substitutions were associated with increased affinity for cap nucleoside by factors of 10 and 50, respectively. VP39 interacted more strongly with a true substrate (containing portions of RNA downstream of the cap in addition to the cap itself) than with isolated cap nucleoside, by several orders of magnitude. VP39 mutants with tryptophan substitution at position 180 exhibited apparent defects in substrate catalytic rate during the first turnover cycle, indicating the possibility of an exquisite sensitivity of the catalytic center to subtle changes in substrate position brought about by alterations in the cap-binding slot. The X-ray structure of VP39 with a genuine nucleobase analogue of N7-methylguanosine, namely, N7,9-dimethylguanine, indicated that the N7-methylguanosine rotational orientation within the stack is a property of the cap nucleobase itself.

Crystallography, X-Ray↗
PubMed2002

Path of an RNA ligand around the surface of the vaccinia VP39 subunit of its cognate VP39-VP55 protein heterodimer.

VP39 is a vaccinia virus-encoded RNA modifying protein with roles in the modification of both mRNA ends. At the 3' end it acts as a processivity factor for the vaccinia poly(A) polymerase (VP55), promoting poly(A) tail elongation. Despite VP39's three-dimensional structure having been elucidated along with details of its mode of mRNA 5' end binding, the VP39-VP55 heterodimer's molecular mechanism of processivity is largely unknown. Here, the area immediately above almost the entire surface of the VP39 subunit was probed using chemical reporters, and the path of a previously unidentified RNA binding site was revealed. The path was indicated to fall within a cleft formed by the intersubunit interface and was consistent with both a previously reported model of the heterodimer-nucleic acid ternary complex and the known function of the heterodimer in processive poly(A) tail elongation.

Binding Sites↗