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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) Fold changes in gene expression for the genes that are less affected in PBS mutants were overlaid onto the fold changes in gene expression of the same genes obtained in the presence of SBS mutants. In these plots, mean values of fold changes of two different PBS mutants (Y80C and F62S) were overlaid onto the mean values obtained from N340S and G324D SBS mutants. Left panel depicts data from coding genes, whereas the right panel is for non-coding regions along the antisense direction. (B) Validation of the microarray data of the selective genes by qRT-PCR. Upper panel shows the microarray profiles of the indicated genes in the presence of Y80C and N340S Rho mutants. Bottom panel validates the expression profiles of the same genes using qRT-PCR. Fold changes in qRT-PCR were expressed in terms of CT values (threshold cycle) as per the convention, details of which are given in the Materials and Methods section. Error bars were calculated from the data obtained from RNA preparations of two independent colonies. Double-sided arrows indicate the differences in gene expression levels in the presence of PBS and SBS mutants.
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Figure 2: (A) Fold changes in gene expression for the genes that are less affected in PBS mutants were overlaid onto the fold changes in gene expression of the same genes obtained in the presence of SBS mutants. In these plots, mean values of fold changes of two different PBS mutants (Y80C and F62S) were overlaid onto the mean values obtained from N340S and G324D SBS mutants. Left panel depicts data from coding genes, whereas the right panel is for non-coding regions along the antisense direction. (B) Validation of the microarray data of the selective genes by qRT-PCR. Upper panel shows the microarray profiles of the indicated genes in the presence of Y80C and N340S Rho mutants. Bottom panel validates the expression profiles of the same genes using qRT-PCR. Fold changes in qRT-PCR were expressed in terms of CT values (threshold cycle) as per the convention, details of which are given in the Materials and Methods section. Error bars were calculated from the data obtained from RNA preparations of two independent colonies. Double-sided arrows indicate the differences in gene expression levels in the presence of PBS and SBS mutants.

Mentions: We performed microarray analyses of MG1655Δrho strain expressing WT and mutant Rho genes from a low copy pCL1920 plasmid and plotted the fold-change in gene expression of different mutants relative to that of the WT (signal intensity with respect to the WT; Figures 1E–H and 2A, Supplementary Figure S5A and S5B). For SBS mutants, MG1655rac− strains (devoid of rac prophage) were used. We observed the following.


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) Fold changes in gene expression for the genes that are less affected in PBS mutants were overlaid onto the fold changes in gene expression of the same genes obtained in the presence of SBS mutants. In these plots, mean values of fold changes of two different PBS mutants (Y80C and F62S) were overlaid onto the mean values obtained from N340S and G324D SBS mutants. Left panel depicts data from coding genes, whereas the right panel is for non-coding regions along the antisense direction. (B) Validation of the microarray data of the selective genes by qRT-PCR. Upper panel shows the microarray profiles of the indicated genes in the presence of Y80C and N340S Rho mutants. Bottom panel validates the expression profiles of the same genes using qRT-PCR. Fold changes in qRT-PCR were expressed in terms of CT values (threshold cycle) as per the convention, details of which are given in the Materials and Methods section. Error bars were calculated from the data obtained from RNA preparations of two independent colonies. Double-sided arrows indicate the differences in gene expression levels in the presence of PBS and SBS mutants.
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Related In: Results  -  Collection

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Figure 2: (A) Fold changes in gene expression for the genes that are less affected in PBS mutants were overlaid onto the fold changes in gene expression of the same genes obtained in the presence of SBS mutants. In these plots, mean values of fold changes of two different PBS mutants (Y80C and F62S) were overlaid onto the mean values obtained from N340S and G324D SBS mutants. Left panel depicts data from coding genes, whereas the right panel is for non-coding regions along the antisense direction. (B) Validation of the microarray data of the selective genes by qRT-PCR. Upper panel shows the microarray profiles of the indicated genes in the presence of Y80C and N340S Rho mutants. Bottom panel validates the expression profiles of the same genes using qRT-PCR. Fold changes in qRT-PCR were expressed in terms of CT values (threshold cycle) as per the convention, details of which are given in the Materials and Methods section. Error bars were calculated from the data obtained from RNA preparations of two independent colonies. Double-sided arrows indicate the differences in gene expression levels in the presence of PBS and SBS mutants.
Mentions: We performed microarray analyses of MG1655Δrho strain expressing WT and mutant Rho genes from a low copy pCL1920 plasmid and plotted the fold-change in gene expression of different mutants relative to that of the WT (signal intensity with respect to the WT; Figures 1E–H and 2A, Supplementary Figure S5A and S5B). For SBS mutants, MG1655rac− strains (devoid of rac prophage) were used. We observed the following.

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