Rho-dependent transcription termination is the dominant mechanism in Mycobacterium tuberculosis.

Intrinsic and Rho-dependent transcription termination mechanisms regulate gene expression and recycle RNA polymerase in bacteria. Both the modes are well studied in Escherichia coli, and a few other organisms. The understanding of Rho function is limited in most other bacteria including mycobacteria. Here, we highlight the dominance of Rho-dependent termination in mycobacteria and validate Rho as a key regulatory factor. The lower abundance of intrinsic terminators, high cellular levels of Rho, and its genome-wide association with a majority of transcriptionally active genes indicate the pronounced role of Rho-mediated termination in Mycobacterium tuberculosis (Mtb). Rho modulates the termination of RNA synthesis for both protein-coding and stable RNA genes in Mtb. Concordantly, the depletion of Rho in mycobacteria impact its growth and enhances the transcription read-through at 3' ends of the transcription units. We demonstrate that MtbRho is catalytically active in the presence of RNA with varied secondary structures. These properties suggest an evolutionary adaptation of Rho as the efficient and preponderant mode of transcription termination in mycobacteria.

Intrinsic and Rho-dependent termination cooperate for efficient transcription termination at 3' untranslated regions.

The intrinsic, and the Rho-dependent mechanisms of transcription termination are conserved in bacteria. Generally, the two mechanisms have been illustrated as two independent pathways occurring in the 3' ends of different genes with contrasting requirements to halt RNA synthesis. However, a majority of intrinsic terminators terminate transcription inefficiently leading to transcriptional read-through. The unwanted transcription in the downstream region beyond the terminator would have undesired consequences. To prevent such transcriptional read-through, bacteria must have evolved ways to terminate transcription more efficiently at or near the termination sites. We describe the participation of both the mechanisms, where intrinsic terminator and Rho factor contribute to prevent transcriptional read-through. Contribution from both the termination processes is demonstrated at the downstream regions of the genes both in vitro and in vivo in mycobacteria. Distinct patterns of cooperation between the two modes of termination were observed at the 3' untranslated regions of the genes to ensure efficient termination. We demonstrate similar mode of operation between the two termination processes in Escherichia coli suggesting a likely prevalence of this cooperation across bacteria. The reporter system developed to assess the Rho - intrinsic termination collaboration in vivo for mycobacteria and E. coli can readily be applied to other bacteria.

Revisiting intrinsic transcription termination in mycobacteria: U-tract downstream of secondary structure is dispensable for termination.

In Escherichia coli, G/C-rich hairpin structure followed by a U-tract in the 3' region of the nascent RNA are crucial determinants for intrinsic or factor independent transcription termination. In mycobacteria, there is a scarcity of such intrinsic terminators. However, secondary structures having G/C-rich stem devoid of any U's or with suboptimal U-tracts were identified earlier as terminators and found to be functional both in vitro and in vivo. Two different observations - that a mycobacterial RNA polymerase (RNAP) does not function at intrinsic terminators devoid of U-tracts and the identification of an altogether new motif for termination in mycobacteria necessitated re-examining a number of putative terminators for their function as terminators. When these in silico identified non-canonical terminators were subjected to experimental validation, they were found to dissociate RNA from the elongating RNAP. Termination is observed when the U-tracts were reduced, or totally absent both in vitro and in vivo. Our results, thus indicate that the presence of U-tract following the G/C-rich stem in an intrinsic terminator may not be an essential determinant for transcription termination in mycobacteria.

Evaluation of Fracture Resistance of Endodontically Treated Teeth Filled with Gutta-Percha and Resilon Obturating Material: An In Vitro Study.

BACKGROUND: As per many studies endodontically treated teeth are widely considered to be more susceptible to fracture than vital teeth. Obturation strains and post placement have been a major cause of vertical root fracture. Present study was conducted to compare in vitro fracture resistance after filling with either Gutta-percha or Resilon by lateral condensation techniques in root canals. This study evaluated a new thermoplastic synthetic polymer based on polyester, which contains bioactive and radiopaque filler, Resilon performs every way as Gutta-percha except that it allows the bonding agent to attach to the resin core and the dentin wall thus forming a monoblock. MATERIALS AND METHODS: In the present study 90 freshly extracted single-rooted human mandibular premolar teeth endodontically treated, were cut at the cemento-enamel junction, and were randomly divided into three groups of 30 each as teeth of Group A (Control) received no obturation, Group B teeth were obturated using Gutta-percha/AH26, and Group C teeth were obturated using Resilon/Epiphany obturating kit. Each specimen were mounted in acrylic in a polyvinyl ring and then tested for fracture resistance with the help of an universal testing machine. A compressive force was applied until the root is fractured. The data were subjected to analysis of variance for comparing mean difference of fracture resistance among three groups. Multiple comparisons among these groups were carried out by non-parametric Kruskal-Wallis analysis. A p value of <0.0001 was considered a statistically significant difference. RESULTS: The results obtain after analysis showed no significant differences in the fracture resistance between the two tested groups of endodontic sealers. CONCLUSION: Within the limitation of the present in-vitro study, Resilon/Epiphany sealer performs better than Gutta-percha/AH 26 sealer with lateral condensation technique.

Management of Class-II Furcation Complicated with Endodontic involvement using Two Different Regenerative Materials.

This paper presents a case series of furcation involved teeth complicated with endodontic involvement which were treated with periodontal, endodontic and restorative procedures using different bone regenerative materials like; (a) Calcium phosphosilicate bone substitute having bioactive glass 69% mixed with glycerin 19% and poly-ethylene 12% dispensed in a putty form; (b) hydroxyapatite 70% and ß-tricalcium phosphate 30% dispensed in granular form. All the cases were randomly selected having Grade II furcation defect with primary or secondary endodontic involvement. All cases were under observation for a period of 9 months. Measurements at 9 months post-surgery demonstrated that dental putty as bone graft substitute which was in combination of bioactive glass mixed with glycerine and polyethylene glycol showed better result as compared granular bone graft which was in combination of hydroxyapatite and ß-tricalcium phosphate.