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Monitoring processivity and length control of a carbohydrate polymerase.

ABSTRACT: Carbohydrate polymerases are abundant in nature. Although they play vital physiological roles, the molecular mechanisms that they use for the controlled assembly of polymers are largely unknown. One fundamental issue is whether an enzyme utilizes a processive or distributive mechanism for chain elongation. The shortage of mechanistic information on polysaccharide-generating glycosyltransferases became apparent when we sought to carry out investigations of GlfT2, a glycosyltransferase essential for cell wall biosynthesis in Mycobacterium tuberculosis. GlfT2 catalyzes the formation of the cell wall galactan, which is a linear polysaccharide consisting of 20-40 repeating d-galactofuranose (Galf) residues. Recombinant GlfT2 can act on synthetic acceptors to produce polymers with lengths similar to those of endogenous galactan, indicating that GlfT2 has an intrinsic ability to control polymer length. To address whether GlfT2 utilizes a processive or distributive mechanism, we developed a mass spectrometry assay. Our approach, which relies on acceptors labeled with stable isotopes, provides direct evidence that GlfT2 is a processive polymerase that maintains contact with the glycan substrate through successive monomer additions. Given this finding, we probed further the catalytic mechanism of GlfT2 to address the basis of an observed kinetic lag phase. These studies suggest that GlfT2 possesses subsites for Galf residue binding and that substrates that can fill these subsites undergo efficient processive polymerization. The presence of these subsites and the kinetic lag phase are common features of processive enzymes. We anticipate that the strategies described herein can be applied to mechanistic studies of other carbohydrate polymerization reactions.

SUBMITTER: Levengood MR

PROVIDER: S-EPMC3179903 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

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Monitoring processivity and length control of a carbohydrate polymerase.

Levengood Matthew R MR Splain Rebecca A RA Kiessling Laura L LL

Journal of the American Chemical Society 20110725 32

Carbohydrate polymerases are abundant in nature. Although they play vital physiological roles, the molecular mechanisms that they use for the controlled assembly of polymers are largely unknown. One fundamental issue is whether an enzyme utilizes a processive or distributive mechanism for chain elongation. The shortage of mechanistic information on polysaccharide-generating glycosyltransferases became apparent when we sought to carry out investigations of GlfT2, a glycosyltransferase essential f ...[more]

PMID: 21739979

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Project description:To determine the role of ICP22 in transcription, we performed precise nuclear run-on followed by deep sequencing (PRO-seq) and global nuclear run-on with sequencing (GRO-seq) in cells infected with a viral mutant lacking the entire ICP22-encoding α22 (US1/US1.5) gene and a virus derived from this mutant bearing a restored α22 gene. At 3 h postinfection (hpi), the lack of ICP22 reduced RNA polymerase (Pol) promoter proximal pausing (PPP) on the immediate early α4, α0, and α27 genes. Diminished PPP at these sites accompanied increased Pol processivity across the entire herpes simplex virus 1 (HSV-1) genome in GRO-seq assays, resulting in substantial increases in antisense and intergenic transcription. The diminished PPP on α gene promoters at 3 hpi was distinguishable from effects caused by treatment with a viral DNA polymerase inhibitor at this time. The ICP22 mutant had multiple defects at 6 hpi, including lower viral DNA replication, reduced Pol activity on viral genes, and increased Pol activity on cellular genes. The lack of ICP22 also increased PPP release from most cellular genes, while a minority of cellular genes exhibited decreased PPP release. Taken together, these data indicate that ICP22 acts to negatively regulate transcriptional elongation on viral genes in part to limit antisense and intergenic transcription on the highly compact viral genome. This regulatory function directly or indirectly helps to retain Pol activity on the viral genome later in infection. IMPORTANCE The longstanding observation that ICP22 reduces RNA polymerase II (Pol II) serine 2 phosphorylation, which initiates transcriptional elongation, is puzzling because this phosphorylation is essential for viral replication. The current study helps explain this apparent paradox because it demonstrates significant advantages in negatively regulating transcriptional elongation, including the reduction of antisense and intergenic transcription. Delays in elongation would be expected to facilitate the ordered assembly and functions of transcriptional initiation, elongation, and termination complexes. Such limiting functions are likely to be important in herpesvirus genomes that are otherwise highly transcriptionally active and compact, comprising mostly short, intronless genes near neighboring genes of opposite sense and containing numerous 3'-nested sets of genes that share transcriptional termination signals but differ at transcriptional start sites on the same template strand.

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Monitoring processivity and length control of a carbohydrate polymerase.

Publications

Monitoring processivity and length control of a carbohydrate polymerase.

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