Category:TeamRed

DNA replication depends on the synthesis of nrdA and nrdB which depends on the synthesis of the gene frd. Figure 1 is the gene map. Figure 6 shows the kinetics of when each of the genes are synthesized. It also shows the fact that nrdA and nrdB synthesis begins after the synthesis of frd.

Both nrdA and nrdB were cleaved out of T4 phage genome. They were then put into a plasmid vector (pnrdAB)and implanted into E. coli N4830. Expression was induced by increasing temperature. Ribonucleoside diphosphate reductase was observed to increase in the E. coli strains containing the pnrdAB vector.

DNA replication depends on the synthesis of nrdA and nrdB which depends on the synthesis of the gene frd. Figure 1 is the gene map. Figure 6 shows the kinetics of when each of the genes are synthesized. It also shows the fact that nrdA and nrdB synthesis begins after the synthesis of frd.

The BLASTp e-values for Troll gene 127 blasted against Bacillus strain 168 gave a value of 4e^-15. When reversed the BLAST from strain 168 to Troll, e-value was 2e^-14. The BLASTp results show orthology for nrdE of Bacillus strain 168.

Addition of 1M guanidine HCL cause dissociation of proteins B1 and B2. B2 protein recovered was enzymatically inactive until treatment with iron. Preparations of B2 containing 2.3g and 2.1g atoms of iron per mol and an excess of active B1 both showed ribonucleotide reductase activity yielded 315nM and 280nM of NADPH respectively. This can be observed in Fig4.

The molecular activity of native reductase is between 200nm and 300nm. Also, solutions containing inactive B2 without iron had 2% the activity of iron treated B2.

This paper follows the studies in another article (PMID 4627024) which I don't have access to read past the beginning results and the abstract. This previous article used a temperature sensitive mutant ts-167 to determine the function of the nrdEF locus. That paper determined the function by analyzing the pool of dNTP present at the restrictive temperature. dCTP and dATP were not apparent at these restrictive temperatures.

The reference listed above (PMID 8969495) used phylogeny and sequencing to determine that Enterobacteria favor the use of nrdAB loci, whereas Bacilus subtils has evolved to use nrdEF. They also used clone pMP25.8 to rescue the temperature sensitive mutant, and later sequenced the nrdE mutant to determine there were 2 neutral mutations and 1 mutation that led to the lethality of the temperature sensitive mutant. They concluded that this mutation was the cause for the loss of function of the NrdE protein.

The mutant lethality due to the loss of dCTP and dATP synthesis provides evidenct that this gene is responsible for the essential enzyme responsible for reductase activity for the respective ribonucleoside-diphosphates.

Inferred homology from protein in Enteriobacteria phage T4. BLASTp results show a match to NrdA, NP_049845.1, with 85% coverage, e-value of 7e-58, and identity of 27%

Figure 4 shows that NrdA and NrdB from bacteriophage T4 have no activity alone; both are necessary to form a complex capable of ribonucleoside-diphosphate reductase activity