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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) Gene organization in the rac prophage region. Location of the trac terminator is indicated. Expression of kilR, an inhibitor of Ftz, is lethal to the cells. In (B), growth of MG1655rac+Δrho::kanR strains in the presence of different rho mutants expressed from a low copy plasmid pCl1920 is shown. Two transductants of each rho mutant from the P1 transduction plates, as shown in Supplementary Figure S4A, were re-streaked on LB plates. (C) and (D) β-galactosidase activities of MC4100 rac−Δrho::kanR strains harboring pCl1920 plasmids expressing different PBS and SBS mutants. In (C), the lacZ reporter cassette was fused downstream of trac terminator, and in (D), it was fused to tR1 terminator. Both the reporters were present as λRS45 lysogens. Error bars were calculated from the values obtained from six independent colonies. Fold changes in the activity with respect to the WT are indicated. (E)–(H) Plots of microarray profiles obtained from MG1655rac+ (for PBS) and MG1655rac− (for SBS) strains expressing different rho mutants as indicated. The ratio of the hybridization intensities obtained from WT and different mutants gave the measure of fold change that is expressed in log2 scale as per convention. ‘+’ fold changes denote up-regulation, whereas ‘–’ fold changes denote down-regulation of the genes. In (I) and (J), expressions of the genes in the rac prophage region were highlighted for the two PBS Rho mutants. Data were taken from (E) and (F).
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Figure 1: (A) Gene organization in the rac prophage region. Location of the trac terminator is indicated. Expression of kilR, an inhibitor of Ftz, is lethal to the cells. In (B), growth of MG1655rac+Δrho::kanR strains in the presence of different rho mutants expressed from a low copy plasmid pCl1920 is shown. Two transductants of each rho mutant from the P1 transduction plates, as shown in Supplementary Figure S4A, were re-streaked on LB plates. (C) and (D) β-galactosidase activities of MC4100 rac−Δrho::kanR strains harboring pCl1920 plasmids expressing different PBS and SBS mutants. In (C), the lacZ reporter cassette was fused downstream of trac terminator, and in (D), it was fused to tR1 terminator. Both the reporters were present as λRS45 lysogens. Error bars were calculated from the values obtained from six independent colonies. Fold changes in the activity with respect to the WT are indicated. (E)–(H) Plots of microarray profiles obtained from MG1655rac+ (for PBS) and MG1655rac− (for SBS) strains expressing different rho mutants as indicated. The ratio of the hybridization intensities obtained from WT and different mutants gave the measure of fold change that is expressed in log2 scale as per convention. ‘+’ fold changes denote up-regulation, whereas ‘–’ fold changes denote down-regulation of the genes. In (I) and (J), expressions of the genes in the rac prophage region were highlighted for the two PBS Rho mutants. Data were taken from (E) and (F).

Mentions: To study the differential growth behavior between the WT and different rho mutants (Figure 1), MG1655rac+strains (having the rac prophage) were at first transformed with WT and different Rho mutants expressed from a low copy number plasmid, pCL1920 (pHYD567), and, subsequently, the chromosomal rho was removed by inserting a kanR cassette (rho::kanR) via P1 transduction (Supplementary Figure S4A). The transductants were re-streaked on the LB plates supplemented with appropriate antibiotics (Figure 1B). In this rho::kanR cassette, first 360 bases of the rho sequence are replaced with the kanamycin resistance gene, and hence rho is linked very tightly with this marker (P1 was made on the strain RS330; see Table 1). We have also confirmed the absence of duplication of rho in all the transductants by PCR using rho specific primers (Supplementary Figure S4B).


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) Gene organization in the rac prophage region. Location of the trac terminator is indicated. Expression of kilR, an inhibitor of Ftz, is lethal to the cells. In (B), growth of MG1655rac+Δrho::kanR strains in the presence of different rho mutants expressed from a low copy plasmid pCl1920 is shown. Two transductants of each rho mutant from the P1 transduction plates, as shown in Supplementary Figure S4A, were re-streaked on LB plates. (C) and (D) β-galactosidase activities of MC4100 rac−Δrho::kanR strains harboring pCl1920 plasmids expressing different PBS and SBS mutants. In (C), the lacZ reporter cassette was fused downstream of trac terminator, and in (D), it was fused to tR1 terminator. Both the reporters were present as λRS45 lysogens. Error bars were calculated from the values obtained from six independent colonies. Fold changes in the activity with respect to the WT are indicated. (E)–(H) Plots of microarray profiles obtained from MG1655rac+ (for PBS) and MG1655rac− (for SBS) strains expressing different rho mutants as indicated. The ratio of the hybridization intensities obtained from WT and different mutants gave the measure of fold change that is expressed in log2 scale as per convention. ‘+’ fold changes denote up-regulation, whereas ‘–’ fold changes denote down-regulation of the genes. In (I) and (J), expressions of the genes in the rac prophage region were highlighted for the two PBS Rho mutants. Data were taken from (E) and (F).
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Figure 1: (A) Gene organization in the rac prophage region. Location of the trac terminator is indicated. Expression of kilR, an inhibitor of Ftz, is lethal to the cells. In (B), growth of MG1655rac+Δrho::kanR strains in the presence of different rho mutants expressed from a low copy plasmid pCl1920 is shown. Two transductants of each rho mutant from the P1 transduction plates, as shown in Supplementary Figure S4A, were re-streaked on LB plates. (C) and (D) β-galactosidase activities of MC4100 rac−Δrho::kanR strains harboring pCl1920 plasmids expressing different PBS and SBS mutants. In (C), the lacZ reporter cassette was fused downstream of trac terminator, and in (D), it was fused to tR1 terminator. Both the reporters were present as λRS45 lysogens. Error bars were calculated from the values obtained from six independent colonies. Fold changes in the activity with respect to the WT are indicated. (E)–(H) Plots of microarray profiles obtained from MG1655rac+ (for PBS) and MG1655rac− (for SBS) strains expressing different rho mutants as indicated. The ratio of the hybridization intensities obtained from WT and different mutants gave the measure of fold change that is expressed in log2 scale as per convention. ‘+’ fold changes denote up-regulation, whereas ‘–’ fold changes denote down-regulation of the genes. In (I) and (J), expressions of the genes in the rac prophage region were highlighted for the two PBS Rho mutants. Data were taken from (E) and (F).
Mentions: To study the differential growth behavior between the WT and different rho mutants (Figure 1), MG1655rac+strains (having the rac prophage) were at first transformed with WT and different Rho mutants expressed from a low copy number plasmid, pCL1920 (pHYD567), and, subsequently, the chromosomal rho was removed by inserting a kanR cassette (rho::kanR) via P1 transduction (Supplementary Figure S4A). The transductants were re-streaked on the LB plates supplemented with appropriate antibiotics (Figure 1B). In this rho::kanR cassette, first 360 bases of the rho sequence are replaced with the kanamycin resistance gene, and hence rho is linked very tightly with this marker (P1 was made on the strain RS330; see Table 1). We have also confirmed the absence of duplication of rho in all the transductants by PCR using rho specific primers (Supplementary Figure S4B).

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