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The RNA-mediated, asymmetric ring regulatory mechanism of the transcription termination Rho helicase decrypted by time-resolved nucleotide analog interference probing (trNAIP).

ABSTRACT: Rho is a ring-shaped, ATP-dependent RNA helicase/translocase that dissociates transcriptional complexes in bacteria. How RNA recognition is coupled to ATP hydrolysis and translocation in Rho is unclear. Here, we develop and use a new combinatorial approach, called time-resolved Nucleotide Analog Interference Probing (trNAIP), to unmask RNA molecular determinants of catalytic Rho function. We identify a regulatory step in the translocation cycle involving recruitment of the 2'-hydroxyl group of the incoming 3'-RNA nucleotide by a Rho subunit. We propose that this step arises from the intrinsic weakness of one of the subunit interfaces caused by asymmetric, split-ring arrangement of primary RNA tethers around the Rho hexamer. Translocation is at highest stake every seventh nucleotide when the weak interface engages the incoming 3'-RNA nucleotide or breaks, depending on RNA threading constraints in the Rho pore. This substrate-governed, 'test to run' iterative mechanism offers a new perspective on how a ring-translocase may function or be regulated. It also illustrates the interest and versatility of the new trNAIP methodology to unveil the molecular mechanisms of complex RNA-based systems.

SUBMITTER: Soares E

PROVIDER: S-EPMC4132721 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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The RNA-mediated, asymmetric ring regulatory mechanism of the transcription termination Rho helicase decrypted by time-resolved nucleotide analog interference probing (trNAIP).

Soares Emilie E Schwartz Annie A Nollmann Marcello M Margeat Emmanuel E Boudvillain Marc M

Nucleic acids research 20140712 14

Rho is a ring-shaped, ATP-dependent RNA helicase/translocase that dissociates transcriptional complexes in bacteria. How RNA recognition is coupled to ATP hydrolysis and translocation in Rho is unclear. Here, we develop and use a new combinatorial approach, called time-resolved Nucleotide Analog Interference Probing (trNAIP), to unmask RNA molecular determinants of catalytic Rho function. We identify a regulatory step in the translocation cycle involving recruitment of the 2'-hydroxyl group of t ...[more]

PMID: 25016524

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Project description:The termination factor Rho, an ATP-dependent RNA translocase, preempts pervasive transcription processes, thereby rendering genome integrity in bacteria. Here, we show that the loss of Rho function raised the intracellular pH to >8.0 in Escherichia coli. The loss of Rho function upregulates tryptophanase-A (TnaA), an enzyme that catabolizes tryptophan to produce indole, pyruvate, and ammonia. We demonstrate that the enhanced TnaA function had produced the conjugate base ammonia, raising the cellular pH in the Rho-dependent termination defective strains. On the other hand, the constitutively overexpressed Rho lowered the cellular pH to about 6.2, independent of cellular ammonia levels. Since Rho overexpression may increase termination activities, the decrease in cellular pH could result from an excess H+ ion production during ATP hydrolysis by overproduced Rho. Furthermore, we performed in vivo termination assays to show that the efficiency of Rho-dependent termination was increased at both acidic and basic pH ranges. Given that the Rho level remained unchanged, the alkaline pH increases the termination efficiency by stimulating Rho's catalytic activity. We conducted the Rho-mediated RNA release assay from a stalled elongation complex to show an efficient RNA release at alkaline pH, compared to the neutral or acidic pH, that supports our in vivo observation. Whereas acidic pH appeared to increase the termination function by elevating the cellular level of Rho. This study is the first to link Rho function to the cellular pH homeostasis in bacteria. IMPORTANCE The current study shows that the loss or gain of Rho-dependent termination alkalizes or acidifies the cytoplasm, respectively. In the case of loss of Rho function, the tryptophanase-A enzyme is upregulated, and degrades tryptophan, producing ammonia to alkalize cytoplasm. We hypothesize that Rho overproduction by deleting its autoregulatory DNA portion increases termination function, causing excessive ATP hydrolysis to produce H+ ions and cytoplasmic acidification. Therefore, this study is the first to unravel a relationship between Rho function and intrinsic cellular pH homeostasis. Furthermore, the Rho level increases in the absence of autoregulation, causing cytoplasmic acidification. As intracellular pH plays a critical role in enzyme function, such a connection between Rho function and alkalization will have far-reaching implications for bacterial physiology.

Dataset Information

The RNA-mediated, asymmetric ring regulatory mechanism of the transcription termination Rho helicase decrypted by time-resolved nucleotide analog interference probing (trNAIP).

Publications

The RNA-mediated, asymmetric ring regulatory mechanism of the transcription termination Rho helicase decrypted by time-resolved nucleotide analog interference probing (trNAIP).

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