The ribosome recycling factor from Mycobacterium tuberculosis has been crystallized. The monoclinic crystals, with 52.5% solvent content, contain one protein molecule in the asymmetric unit.

The ribosome recycling factor from Mycobacterium tuberculosis has been crystallized. The monoclinic crystals, with 52.5% solvent content, contain one protein molecule in the asymmetric unit.

Ribosome recycling factor (RRF), coded for by the frr locus, is involved in the disassembly of post-termination complexes and recycling of the ribosomes for a fresh round of initiation in bacteria and in eukaryotic organelles. In a cross-species-complementation experiment, it was shown that the Thermus thermophilus RRF protein lacking five amino acids from its C-terminal end (DeltaC5TthRRF) but not the full-length protein (TthRRF) complemented Escherichia, coli for its frr(ts) phenotype. It was also shown that the Mycobacterium tuberculosis RFF protein (MtuRRF) did not complement E. coli LJ14 for frr(ts). However, simultaneous expression of elongation factor G (EFG) and RRF from M. tuberculosis resulted in complementation of E. coli LJ14. Here it is shown that unlike DeltaC5TthRRF, an equivalent mutant of MtuRRF lacking six amino acids from its C-terminal end (DeltaC6MtuRRF) did not complement E. coli LJ14. Surprisingly, DeltaC6MtuRRF failed to complement the strain even in the presence of homologous EFG (MtuEFG). The biochemical and biophysical characterization of these proteins suggested that the mutant RRF folded properly. However, ribosome-binding assays showed that the mutant protein was compromised in its binding to E. coli ribosomes. It is suggested that the conserved amino acids at the C-terminal end of the RRFs contribute to their residency on ribosomes and that the specific interactions between RRF and EFG are crucial in the disassembly of the termination complex.

The binding of three analogues of the minor-groove binding antiviral antibiotic distamycin (Dst) with double-stranded (ds)-DNA were monitored using ds-DNA melting temperature (Tm) measurements, ethidium bromide (EtBr) displacement assay, footprinting analysis and induced circular dichroism (ICD). These compounds contained 3-5 N-methyl-pyrrole-carboxamide units and lacked the N-terminal formamide unit present in Dst. These experiments suggested that the present analogues did not compromise their AT-specificity despite the deletion of the N-terminal formamide unit. The binding affinities, however, were significantly affected. Interestingly, the analogue with three N-methyl-pyrrole-carboxamide units exhibited an initial decrease in ICD at > 40 mM salt concentrations. This was followed by a pronounced recovery of ICD at > 1.6 M salt concentrations, a phenomenon hitherto not observed with any other DNA binding molecules. The pentapyrrole analogue exhibited the highest binding affinity with CT-DNA under normal (40 mM) salt conditions. However, it suffered maximum relative dissociation under high salt conditions and did not exhibit any recovery in ICD at higher NaCl concentrations. The analogues possessing four and five pyrrole rings exhibited intense ICD signals with poly d(GC) in the ligand absorption region in the presence of 40 mM NaCl, unlike the one with three pyrrole rings. These ICD signals were however, highly susceptible to changes in ionic strength. Thus subtle modifications in the ligand molecular structure can have dramatic effect on their DNA binding properties.

Once the translating ribosomes reach a termination codon, the nascent polypeptide chain is released in a factor‐dependent manner. However, the P‐site‐bound deacylated tRNA and the ribosomes themselves remain bound to the mRNA (post‐termination complex). The ribosome recycling factor (RRF) plays a vital role in dissociating this complex. Here we show that the Mycobacterium tuberculosis RRF (MtuRRF) fails to rescue Escherichia coli LJ14, a strain temperature‐sensitive for RRF (frrts). More interestingly, co‐expression of M.tuberculosis elongation factor G (MtuEFG) with MtuRRF rescues the frrts strain of E.coli. The simultaneous expression of MtuEFG is also needed to cause an enhanced release of peptidyl‐tRNAs in E.coli by MtuRRF. These observations provide the first genetic evidence for a functional interaction between RRF and EFG. Both the in vivo and in vitro analyses suggest that RRF does not distinguish between the translating and terminating ribosomes for their dissociation from mRNA. In addition, complementation of E.coli PEM100 (fusAts) with MtuEFG suggests that the mechanism of RRF function is independent of the translocation activity of EFG.

The use of teeth as an important resource in the analysis of forensic case history by polymerase chain reaction (PCR) or other related methods has been reported. However, a major drawback in using teeth has been that the DNA is present only in trace amounts, and the methods to recover DNA from the flinty material have not been efficient or cost effective. In this report, we describe a method to prepare DNA from the hard tooth tissues. Our studies show that ultrasonication of teeth samples yields sufficient amounts of good quality DNA useful for PCR-based diagnostic methods. The teeth could serve as a reliable source of DNA for amplification-based forensic methods in sex determination. DNA could be obtained from any tooth, regardless of the age of subject. Furthermore, by using the AMEL gene-based primers in PCR, we have shown that the AMEL gene serves as a good marker for sex determination in the Indian population. In our study, the PCR-based method was sensitive and proved to be successful for sex determination with a complete specificity.