Polynucleotide phosphorylases from thermophiles

A purification procedure was developed for Eschericia coli polynucleotide phosphorylase, and subsequently applied to polynucleotide phosphorylases from Thermus aquaticus and Bacillus stearothermophilus. Preliminary investigations of the catalytic properties of the thermostable polynucleotide phosphorylases were carried out in the hope of effecting the facile polymerisation of modified nucleotide diphosphates which have a predominantly syn conformation. However, even at elevated temperatures, where the relative proportion of substrate molecules in the anti-conformation may be increased, the specificity of the thermostable enzymes was no broader than that reported for mesophylic enzymes. Other catalytic properties investigated were also similar to those observed using polynucleotide phosphorylases from other sources. Structural studies of the enzyme from B. stearothermophilus revealed a similar gross amino acid composition and molecular weight to the E. coli enzyme. The quaternary structure differs from other polynucleotide phosphorylases in that four apparently identical subunits were identified on polyacrylamide gel electrophoresis under denaturing conditions. The subunits have a molecular weight of 51,000 daltons. Suberimidate cross-linking experiments confirmed a tetrameric structure for the native enzyme. Partially purified polynucleotide phosphorylase from T. aquaticus had a molecular weight of more than 400,000 daltons as judged by gel filtration. Using a 3' exonuclease from Krebs ascites cells to degrade the rapidly labelled giant nuclear RNA from SV 40 transformed mouse cells, the location of virus specific sequences was investigated by hybridisation to purified SV 40 DNA. An apparent enrichment of virus sequences with increasing degradation of the RNA molecules suggests that virus sequences are absent at the 3’ end of giant nuclear RNA

Characterization of the \u27glutamate effect\u27 on the solution thermodynamics and function of the large fragments of the type I DNA polymerases from E.coli and T.aquaticus

In this study, it is shown that the large fragments of the type I DNA polymerase from E.coli (Klenow) and T.aquaticus (Klentaq) display enhanced DNA binding affinity in glutamate vs. chloride. Across the relatively narrow salt concentration ranges often used to obtain salt linkage data, Klenow also displays an apparently decreased linked ion released (¦¤nions) in Kglutamate vs. KCl while Klentaq does not display such an effect. The osmotic stress technique reveals that Klenow and Klentaq DNA binding is associated with the release of ~500 to 600 waters in KCl. For both proteins, replacing chloride with glutamate results in a 70% reduction in the hydration change upon DNA binding (to ~150-200), highlighting glutamate\u27s osmotic role. To further examine this osmotic effect of glutamate, the salt-DNA binding linkages were extended up to 2.5 M Kglutamate. Consequently, a reversal of the salt linkage is observed above 800mM for both proteins. Salt addition titrations confirmed that rebinding of salt displaced polymerase to DNA occurs beyond 1M [Kglutamate]. Non linear analysis of the biphasic salt linkage indicates that the osmotic role of glutamate is responsible for the reversed linkage and allows the quantitative dissection of the ionic and osmotic behaviors. The similar effect of glutamate on the two polymerases results in a relatively constant affinity difference (¦¤¦¤Gobind(KLN-KTQ)¡Ö-3kcal/mol) throughout the entire salt range. The catalytic activity of both polymerases persists into higher [Kglutamate] than [KCl]. However, the re-association of the proteins on the DNA in high Kglutamate does not result in enhanced catalytic activity. These data represent only the second documentation of an apparent reversed salt linkage for a protein-DNA interaction. This unusual behavior is quantitatively accounted for by a shifting balance of ionic and osmotic effects of the glutamate anion

The E.coli RNA degradosome analysis of molecular chaperones and enolase

Normal mRNA turnover is essential for genetic regulation within cells. The E. coli RNA degradosome, a large multi-component protein complex which originates through specific protein interactions, has been referred to as the “RNA decay machine” and is responsible for mRNA turnover. The degradosome functions to process RNA and its key components have been identified. The scaffold protein is RNase E and it tethers the degradosome to the cytoplasmic membrane. Polynucleotide phosphorylase (PNPase), ATP-dependent RNA helicase (RhlB helicase) and the glycolytic enzyme enolase associate with RNase E to form the degradosome. Polyphosphate kinase associates with the degradosome in substoichiometric amounts, as do the molecular chaperones DnaK and GroEL. The role of DnaK as well as that of enolase in the RNA degradosome is unknown. Very limited research has been conducted on the components of the RNA degradosome under conditions of stress. The aim of this study was to understand the role played by enolase in the assembly of the degradosome under conditions of stress, as well as investigating the protein levels of molecular chaperones under these conditions. The RNA degradosome was successfully purified through its scaffold protein using nickel-affinity chromatography. In vivo studies were performed to investigate the protein levels of DnaK and GroEL present in the degradosome under conditions of heat stress, and whether GroEL could functionally replace DnaK in the degradosome. To investigate the recruitment of enolase to the degradosome under heat stress, a subcellular fractionation was performed to determine the localization of enolase upon heat shock in vivo. The elevated temperature resulted in an increased concentration of enolase in the membrane fraction. To determine whether there is an interaction between enolase and DnaK, enolase activity assays were conducted in vitro. The effect of DnaK on enolase activity was measured upon quantifying DnaK and adding it to the enolase assays. For the first time it was observed that the activity of enolase increased with the addition of substoichiometric amounts of DnaK. This indicates that DnaK may be interacting with the RNA degradosome via enolase

ENZYMES: Catalysis, Kinetics and Mechanisms

Onemarvelsattheintricate designoflivingsystems,andwecannotbutwonderhow life originated on this planet. Whether ?rst biological structures emerged as the selfreproducing genetic templates (genetics-?rst origin of life) or the metabolic universality preceded the genome and eventually integrated it (metabolism-?rst origin of life) is still a matter of hot scienti?c debate. There is growing acceptance that the RNA world came ?rst – as RNA molecules can perform both the functions of information storage and catalysis. Regardless of which view eventually gains acceptance, emergence of catalytic phenomena is at the core of biology. The last century has seen an explosive growth in our understanding of biological systems. The progression has involved successive emphasis on taxonomy ! physiology ! biochemistry ! molecular biology ! genetic engineering and ?nally the large-scale study of genomes. The ?eld of molecular biology became largely synonymous with the study of DNA – the genetic material. Molecular biology however had its beginnings in the understanding of biomolecular structure and function. Appreciationofproteins,catalyticphenomena,andthefunctionofenzymeshadalargeroleto play in the progress of modern biology