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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) Schematic showing the design of the formation of stalled elongation complexes (RB) downstream of the rut sites of either H-19B tR1 or trac terminators using lac repressor. Different components of the quaternary complex have been indicated. The length of the nascent RNA is 374 nt and 435 nt for RBs with tR1 and trac terminators, respectively. (B) and (C) Autoradiogram showing the Rho-induced RNA release kinetics from the RBs formed downstream of indicated terminator sequences both in the absence and presence of NusG. ‘S’ denotes half of the supernatant and ‘P’ denotes rest of the sample. RB denotes the RNA corresponding to the position of the stalled EC and RO indicates the run-off product of this template. (D) and (E) Fractions of released RNA in the above experiments were plotted against time for the indicated RB complexes formed downstream of the two terminators. Plots obtained in the absence (-•-) and presence of (-o-) NusG are indicated. The experimental data points were fitted either to an exponential rise form or to a logistic function as described in the Materials and Methods section. The delay in the initiation of RNA release for trac has been indicated by a flat slope drawn as a dashed line. Rates of RNA release of both the curves are indicated by the slopes drawn as solid lines. Note that the rate estimated for −NusG curve is from the incremental phase following the lag phase of the curve. (F) Fraction of ATP hydrolyzed by Rho was plotted against time. These reactions were induced by the nascent RNA from the stalled ECs (as described in Figure 4A) formed downstream of the indicated terminators. A significant lag to initiate the ATP hydrolysis was observed at the trac terminator. An amplified version of this lag is shown in the inset.
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Figure 4: (A) Schematic showing the design of the formation of stalled elongation complexes (RB) downstream of the rut sites of either H-19B tR1 or trac terminators using lac repressor. Different components of the quaternary complex have been indicated. The length of the nascent RNA is 374 nt and 435 nt for RBs with tR1 and trac terminators, respectively. (B) and (C) Autoradiogram showing the Rho-induced RNA release kinetics from the RBs formed downstream of indicated terminator sequences both in the absence and presence of NusG. ‘S’ denotes half of the supernatant and ‘P’ denotes rest of the sample. RB denotes the RNA corresponding to the position of the stalled EC and RO indicates the run-off product of this template. (D) and (E) Fractions of released RNA in the above experiments were plotted against time for the indicated RB complexes formed downstream of the two terminators. Plots obtained in the absence (-•-) and presence of (-o-) NusG are indicated. The experimental data points were fitted either to an exponential rise form or to a logistic function as described in the Materials and Methods section. The delay in the initiation of RNA release for trac has been indicated by a flat slope drawn as a dashed line. Rates of RNA release of both the curves are indicated by the slopes drawn as solid lines. Note that the rate estimated for −NusG curve is from the incremental phase following the lag phase of the curve. (F) Fraction of ATP hydrolyzed by Rho was plotted against time. These reactions were induced by the nascent RNA from the stalled ECs (as described in Figure 4A) formed downstream of the indicated terminators. A significant lag to initiate the ATP hydrolysis was observed at the trac terminator. An amplified version of this lag is shown in the inset.

Mentions: (A) Schematic showing the design of the formation of stalled elongation complexes (RB) downstream of the rut sites of either H-19B tR1 or trac terminators using lac repressor. Different components of the quaternary complex have been indicated. The length of the nascent RNA is 374 nt and 435 nt for RBs with tR1 and trac terminators, respectively. (B) and (C) Autoradiogram showing the Rho-induced RNA release kinetics from the RBs formed downstream of indicated terminator sequences both in the absence and presence of NusG. ‘S’ denotes half of the supernatant and ‘P’ denotes rest of the sample. RB denotes the RNA corresponding to the position of the stalled EC and RO indicates the run-off product of this template. (D) and (E) Fractions of released RNA in the above experiments were plotted against time for the indicated RB complexes formed downstream of the two terminators. Plots obtained in the absence (-•-) and presence of (-o-) NusG are indicated. The experimental data points were fitted either to an exponential rise form or to a logistic function as described in the Materials and Methods section. The delay in the initiation of RNA release for trac has been indicated by a flat slope drawn as a dashed line. Rates of RNA release of both the curves are indicated by the slopes drawn as solid lines. Note that the rate estimated for −NusG curve is from the incremental phase following the lag phase of the curve. (F) Fraction of ATP hydrolyzed by Rho was plotted against time. These reactions were induced by the nascent RNA from the stalled ECs (as described in Figure 4A) formed downstream of the indicated terminators. A significant lag to initiate the ATP hydrolysis was observed at the trac terminator. An amplified version of this lag is shown in the inset.


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) Schematic showing the design of the formation of stalled elongation complexes (RB) downstream of the rut sites of either H-19B tR1 or trac terminators using lac repressor. Different components of the quaternary complex have been indicated. The length of the nascent RNA is 374 nt and 435 nt for RBs with tR1 and trac terminators, respectively. (B) and (C) Autoradiogram showing the Rho-induced RNA release kinetics from the RBs formed downstream of indicated terminator sequences both in the absence and presence of NusG. ‘S’ denotes half of the supernatant and ‘P’ denotes rest of the sample. RB denotes the RNA corresponding to the position of the stalled EC and RO indicates the run-off product of this template. (D) and (E) Fractions of released RNA in the above experiments were plotted against time for the indicated RB complexes formed downstream of the two terminators. Plots obtained in the absence (-•-) and presence of (-o-) NusG are indicated. The experimental data points were fitted either to an exponential rise form or to a logistic function as described in the Materials and Methods section. The delay in the initiation of RNA release for trac has been indicated by a flat slope drawn as a dashed line. Rates of RNA release of both the curves are indicated by the slopes drawn as solid lines. Note that the rate estimated for −NusG curve is from the incremental phase following the lag phase of the curve. (F) Fraction of ATP hydrolyzed by Rho was plotted against time. These reactions were induced by the nascent RNA from the stalled ECs (as described in Figure 4A) formed downstream of the indicated terminators. A significant lag to initiate the ATP hydrolysis was observed at the trac terminator. An amplified version of this lag is shown in the inset.
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Related In: Results  -  Collection

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Figure 4: (A) Schematic showing the design of the formation of stalled elongation complexes (RB) downstream of the rut sites of either H-19B tR1 or trac terminators using lac repressor. Different components of the quaternary complex have been indicated. The length of the nascent RNA is 374 nt and 435 nt for RBs with tR1 and trac terminators, respectively. (B) and (C) Autoradiogram showing the Rho-induced RNA release kinetics from the RBs formed downstream of indicated terminator sequences both in the absence and presence of NusG. ‘S’ denotes half of the supernatant and ‘P’ denotes rest of the sample. RB denotes the RNA corresponding to the position of the stalled EC and RO indicates the run-off product of this template. (D) and (E) Fractions of released RNA in the above experiments were plotted against time for the indicated RB complexes formed downstream of the two terminators. Plots obtained in the absence (-•-) and presence of (-o-) NusG are indicated. The experimental data points were fitted either to an exponential rise form or to a logistic function as described in the Materials and Methods section. The delay in the initiation of RNA release for trac has been indicated by a flat slope drawn as a dashed line. Rates of RNA release of both the curves are indicated by the slopes drawn as solid lines. Note that the rate estimated for −NusG curve is from the incremental phase following the lag phase of the curve. (F) Fraction of ATP hydrolyzed by Rho was plotted against time. These reactions were induced by the nascent RNA from the stalled ECs (as described in Figure 4A) formed downstream of the indicated terminators. A significant lag to initiate the ATP hydrolysis was observed at the trac terminator. An amplified version of this lag is shown in the inset.
Mentions: (A) Schematic showing the design of the formation of stalled elongation complexes (RB) downstream of the rut sites of either H-19B tR1 or trac terminators using lac repressor. Different components of the quaternary complex have been indicated. The length of the nascent RNA is 374 nt and 435 nt for RBs with tR1 and trac terminators, respectively. (B) and (C) Autoradiogram showing the Rho-induced RNA release kinetics from the RBs formed downstream of indicated terminator sequences both in the absence and presence of NusG. ‘S’ denotes half of the supernatant and ‘P’ denotes rest of the sample. RB denotes the RNA corresponding to the position of the stalled EC and RO indicates the run-off product of this template. (D) and (E) Fractions of released RNA in the above experiments were plotted against time for the indicated RB complexes formed downstream of the two terminators. Plots obtained in the absence (-•-) and presence of (-o-) NusG are indicated. The experimental data points were fitted either to an exponential rise form or to a logistic function as described in the Materials and Methods section. The delay in the initiation of RNA release for trac has been indicated by a flat slope drawn as a dashed line. Rates of RNA release of both the curves are indicated by the slopes drawn as solid lines. Note that the rate estimated for −NusG curve is from the incremental phase following the lag phase of the curve. (F) Fraction of ATP hydrolyzed by Rho was plotted against time. These reactions were induced by the nascent RNA from the stalled ECs (as described in Figure 4A) formed downstream of the indicated terminators. A significant lag to initiate the ATP hydrolysis was observed at the trac terminator. An amplified version of this lag is shown in the inset.

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