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Redundancy of primary RNA-binding functions of the bacterial transcription terminator Rho.

Shashni R, Qayyum MZ, Vishalini V, Dey D, Sen R - Nucleic Acids Res. (2014)

Bottom Line: The bacterial transcription terminator, Rho, terminates transcription at half of the operons.These terminators function optimally only through a NusG-assisted recruitment and activation of Rho.Our data also indicate that at these terminators, Rho-EC-bound NusG interaction facilitates the isomerization of Rho into a translocase-competent form by stabilizing the interactions of mRNA with the secondary RNA binding site, thereby overcoming the defects of the primary RNA binding functions.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Transcription, Center for DNA Fingerprinting and Diagnostics, Tuljaguda Complex, 4-1-714 Mozamjahi Road, Nampally, Hyderabad 500001, India.

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A kinetic scheme showing different steps in the Rho-recruitment process. OH and CH denote the ‘open’ and ‘close’ hexamer states of Rho as described in the two crystal structures (25,26). It is likely that the major isomerization step as well as the rate-limiting step involves the OH to CH conversion, which is induced by threading of the RNA into the SBS and by the binding of ATP. Rho translocation events denoted as ‘→→→’ are accompanied by sequential ATP hydrolysis steps. The rate constants defining the binding steps are also indicated. Proposed involvement of NusG at the isomerization step is shown.
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Figure 5: A kinetic scheme showing different steps in the Rho-recruitment process. OH and CH denote the ‘open’ and ‘close’ hexamer states of Rho as described in the two crystal structures (25,26). It is likely that the major isomerization step as well as the rate-limiting step involves the OH to CH conversion, which is induced by threading of the RNA into the SBS and by the binding of ATP. Rho translocation events denoted as ‘→→→’ are accompanied by sequential ATP hydrolysis steps. The rate constants defining the binding steps are also indicated. Proposed involvement of NusG at the isomerization step is shown.

Mentions: Upon comparison of the distance between the Rho-loading regions and the termination zones of different terminators (Supplementary Figures S11A and B; compare the termination zones of in vitro transcription assays), we noticed that the mean distances of the termination zones for trac and tyag (trac-like terminators) are unusually long compared to the tR1-like terminators (Supplementary Figure S11; ∼385–425 nt versus 70–145 nt of the classical terminators). Using in vivo termination assays, we have also confirmed that for trac the termination region is indeed located inside the racR (Supplementary Figure S12). These observations can be interpreted as much longer time requirements of Rho on these two terminators to dislodge the EC. Assuming that the translocation speed of Rho remains comparable on different RNA sequences, the following two parameters should determine the aforementioned distance as well as the time taken to dissociate the EC: the time taken to form a translocase-competent Rho at the rut site and the time taken to dislodge the EC. The latter parameter depends on the nature of the EC (20), and hence, following the Rho-loading, isomerization step(s) of forming a translocase-competent Rho is likely to regulate the rate of the whole process (see Figure 5). This theoretical framework predicts that the rate(s) of isomerization step leading to the formation of a translocase-competent form at trac-like terminators is slower as compared to that of the tR1-like terminators. In this context, we also propose that the distance between the rut site and the termination zone can be used to determine the strength of a Rho-dependent terminator.


Redundancy of primary RNA-binding functions of the bacterial transcription terminator Rho.

Shashni R, Qayyum MZ, Vishalini V, Dey D, Sen R - Nucleic Acids Res. (2014)

A kinetic scheme showing different steps in the Rho-recruitment process. OH and CH denote the ‘open’ and ‘close’ hexamer states of Rho as described in the two crystal structures (25,26). It is likely that the major isomerization step as well as the rate-limiting step involves the OH to CH conversion, which is induced by threading of the RNA into the SBS and by the binding of ATP. Rho translocation events denoted as ‘→→→’ are accompanied by sequential ATP hydrolysis steps. The rate constants defining the binding steps are also indicated. Proposed involvement of NusG at the isomerization step is shown.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150792&req=5

Figure 5: A kinetic scheme showing different steps in the Rho-recruitment process. OH and CH denote the ‘open’ and ‘close’ hexamer states of Rho as described in the two crystal structures (25,26). It is likely that the major isomerization step as well as the rate-limiting step involves the OH to CH conversion, which is induced by threading of the RNA into the SBS and by the binding of ATP. Rho translocation events denoted as ‘→→→’ are accompanied by sequential ATP hydrolysis steps. The rate constants defining the binding steps are also indicated. Proposed involvement of NusG at the isomerization step is shown.
Mentions: Upon comparison of the distance between the Rho-loading regions and the termination zones of different terminators (Supplementary Figures S11A and B; compare the termination zones of in vitro transcription assays), we noticed that the mean distances of the termination zones for trac and tyag (trac-like terminators) are unusually long compared to the tR1-like terminators (Supplementary Figure S11; ∼385–425 nt versus 70–145 nt of the classical terminators). Using in vivo termination assays, we have also confirmed that for trac the termination region is indeed located inside the racR (Supplementary Figure S12). These observations can be interpreted as much longer time requirements of Rho on these two terminators to dislodge the EC. Assuming that the translocation speed of Rho remains comparable on different RNA sequences, the following two parameters should determine the aforementioned distance as well as the time taken to dissociate the EC: the time taken to form a translocase-competent Rho at the rut site and the time taken to dislodge the EC. The latter parameter depends on the nature of the EC (20), and hence, following the Rho-loading, isomerization step(s) of forming a translocase-competent Rho is likely to regulate the rate of the whole process (see Figure 5). This theoretical framework predicts that the rate(s) of isomerization step leading to the formation of a translocase-competent form at trac-like terminators is slower as compared to that of the tR1-like terminators. In this context, we also propose that the distance between the rut site and the termination zone can be used to determine the strength of a Rho-dependent terminator.

Bottom Line: The bacterial transcription terminator, Rho, terminates transcription at half of the operons.These terminators function optimally only through a NusG-assisted recruitment and activation of Rho.Our data also indicate that at these terminators, Rho-EC-bound NusG interaction facilitates the isomerization of Rho into a translocase-competent form by stabilizing the interactions of mRNA with the secondary RNA binding site, thereby overcoming the defects of the primary RNA binding functions.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Transcription, Center for DNA Fingerprinting and Diagnostics, Tuljaguda Complex, 4-1-714 Mozamjahi Road, Nampally, Hyderabad 500001, India.

Show MeSH
Related in: MedlinePlus