A nucleotide-switch mechanism mediates opposing catalytic activities of Rel enzymes

Bifunctional Rel stringent factors, the most abundant class of RelA/SpoT homologs, are ribosome-associated enzymes that transfer a pyrophosphate from ATP onto the 3 ' of guanosine tri-/diphosphate (GTP/GDP) to synthesize the bacterial alarmone (p)ppGpp, and also catalyze the 3 ' pyrophosphate hydrolysis to degrade it. The regulation of the opposing activities of Rel enzymes is a complex allosteric mechanism that remains an active research topic despite decades of research. We show that a guanine-nucleotide-switch mechanism controls catalysis by Thermus thermophilus Rel (Rel(Tt)). The binding of GDP/ATP opens the N-terminal catalytic domains (NTD) of Rel(Tt) (Rel(Tt)(NTD)) by stretching apart the two catalytic domains. This activates the synthetase domain and allosterically blocks hydrolysis. Conversely, binding of ppGpp to the hydrolase domain closes the NTD, burying the synthetase active site and precluding the binding of synthesis precursors. This allosteric mechanism is an activity switch that safeguards against futile cycles of alarmone synthesis and degradation

Influência do teor de Mo na microestrutura de ligas Fe-9Cr-xMo Effect of the content of molybdenum in the microstructure of Fe-9Cr-xMo alloy

Aços Cr-Mo são usados na indústria do petróleo em aplicações com óleos crus ricos em compostos sulfurosos. Aços comerciais como 2.5Cr1Mo ou 9Cr1Mo têm se mostrado ineficientes em consequência de altos índices de corrosão naftênica. Uma estratégia para resolver este problema é o aumento do teor de molibdênio destes aços. Neste trabalho foi estudado o efeito do aumento do teor de molibdênio na microestrutura de ligas Fe-9Cr-xMo, solubilizadas e soldadas. Foram levantados os diagramas de fases com auxílio de um programa comercial para verificar as possíveis fases a serem formadas e identificar os problemas de soldagem. A microestrutura das ligas solubilizadas foi analisada por microscopia óptica e EBSD, além da medição da dureza. Foram realizadas soldagens autógenas para verificar o efeito do aporte térmico na microestrutura e na dureza das ligas. O aumento do teor de molibdênio resultou no aumento da dureza das ligas. A análise microestrutural das ligas soldadas apresentou uma particularidade para a liga com menor teor de molibdênio, a presença de martensita. Já as ligas com maior teor de molibdênio apresentaram uma microestrutura completamente ferrítica. A formação de martensita pode ser um problema na solda da liga com menor teor de molibdênio, uma vez que a mesma pode causar perdas nas propriedades mecânicas comprometendo sua aplicação.<br>Cr-Mo steels are used in the petroleum industry in applications with crude oils rich in sulfur compounds. 2.5Cr1Mo or 9Cr1Mo do not resist to operating conditions when in contact with crude oils. The increasing of molybdenum content can improve the corrosion resistance of these alloys. This paper studied the effect of increased concentration of molybdenum in the microstructure of Fe-9Cr-xMo alloys, annealed and welded. Phase diagrams were built with the aid of commercial program to check the possible phases to be formed and to identify the problems of welding. Analyses were realized by optical microscopy, EBSD and hardness tests. Autogenous welds were carried out to verify the effect of heat input on microstructure and hardness of the alloys. The results indicated that the increase in molybdenum concentration resulted in increased hardness of the alloys. After welding the alloy with lower molybdenum content presented the formation of martensite. Alloys with molybdenum content above 5% presented a fully ferritic microstructure. The formation of martensite can be a problem in weld alloys with lower content of molybdenum, since it can cause loss in mechanical properties hindering their application

Comparison of ΔrelA Strains of Escherichia coli and Salmonella enterica Serovar Typhimurium Suggests a Role for ppGpp in Attenuation Regulation of Branched-Chain Amino Acid Biosynthesis

The growth recovery of Escherichia coli K-12 and Salmonella enterica serovar Typhimurium ΔrelA mutants were compared after nutritional downshifts requiring derepression of the branched-chain amino acid pathways. Because wild-type E. coli K-12 and S. enterica serovar Typhimurium LT2 strains are defective in the expression of the genes encoding the branch point acetohydroxy acid synthetase II (ilvGM) and III (ilvIH) isozymes, respectively, ΔrelA derivatives corrected for these mutations were also examined. Results indicate that reduced expression of the known global regulatory factors involved in branched-chain amino acid biosynthesis cannot completely explain the observed growth recovery defects of the ΔrelA strains. In the E. coli K-12 MG1655 ΔrelA background, correction of the preexisting rph-1 allele which causes pyrimidine limitations resulted in complete loss of growth recovery. S. enterica serovar Typhimurium LT2 ΔrelA strains were fully complemented by elevated basal ppGpp levels in an S. enterica serovar Typhimurium LT2 ΔrelA spoT1 mutant or in a strain harboring an RNA polymerase mutation conferring a reduced RNA chain elongation rate. The results are best explained by a dependence on the basal levels of ppGpp, which are determined by relA-dependent changes in tRNA synthesis resulting from amino acid starvations. Expression of the branched-chain amino acid operons is suggested to require changes in the RNA chain elongation rate of the RNA polymerase, which can be achieved either by elevation of the basal ppGpp levels or, in the case of the E. coli K-12 MG1655 strain, through pyrimidine limitations which partially compensate for reduced ppGpp levels. Roles for ppGpp in branched-chain amino acid biosynthesis are discussed in terms of effects on the synthesis of known global regulatory proteins and current models for the control of global RNA synthesis by ppGpp