Biomedical subjects

H E McElroy

Publications and source records attributed to H E McElroy.

3 recordsLinked to original sources

PubMed1995

pTRIPLEscript: a novel cloning vector for generating in vitro transcripts from tandem promoters for SP6, T7 and T3 RNA polymerase.

We have constructed a family of novel in vitro transcription vectors in which functional T3, T7 and SP6 RNA polymerase promoters are arranged in tandem and directed towards a multiple cloning site. This prototype vector, named pTRIPLEscript, permits the transcription of one strand of a DNA insert by any of the three commonly used bacteriophage RNA polymerases with no apparent cross talk, i.e., use of the wrong promoter sequence. The vector has two main uses: (i) to clone probe sequences that will be distributed to many laboratories, allowing the use of the most convenient RNA polymerase; and (ii) to circumvent the problem of RNA polymerase-dependent premature termination.

Base Sequence↗

PubMed1994

Structure of human rhinovirus 3C protease reveals a trypsin-like polypeptide fold, RNA-binding site, and means for cleaving precursor polyprotein.

The structure of human rhinovirus-14 3C protease (3Cpro) has been determined at 2.3 A resolution and refined to an R factor of 0.22. This cysteine protease folds into two topologically equivalent six-stranded beta barrels and in this sense is similar to trypsin-like serine proteases. However, there are differences in the lengths and positioning of individual beta strands as well as in loops connecting elements of secondary structure. The catalytic residues Cys-146, His-40, and Glu-71 are positioned as in serine proteases, but the oxyanion hole is moved 1-1.2 A away. Residues that bind to the 5' noncoding region of rhinovirus genomic RNA are located on the opposite side of the molecule from the active site. Interactions between individual 3Cpro molecules in the crystal lattice suggest a model for intermolecular proteolytic cleavage of the 3CD polyprotein.

3C Viral Proteases↗

PubMed1989

Site-directed alteration of Glu197 and Glu66 in a pyruvoyl-dependent histidine decarboxylase.

Site-directed mutagenesis has been used to explore the role of two carboxylates in the active site of histidine decarboxylase from Lactobacillus 30a. The most striking observation is that conversion of Glu197 to either Gln or Asp causes a major decrease in catalytic rate while enhancing substrate binding. This is consistent with models based on X-ray diffraction results which suggest that the acid may protonate a reaction intermediate during catalysis. The Asp197 protein undergoes a suicide reaction with substrate, apparently triggered by inappropriate protonation of the intermediate. This leads to decarboxylation-dependent transamination which converts the pyruvoyl cofactor to an alanine, inactivating the enzyme. Conversion of Glu66 to Gln affects parameters of kinetic cooperativity. The mutation fixes the Hill number at approximately 1.5, midway between the pH-dependent values of the wild-type enzyme.

Binding Sites↗