The Helicase Activity of Ribonuclease R Is Essential for Efficient Nuclease Activity

Hossain, Sk Tofajjen; Malhotra, Arun; Deutscher, Murray P.

doi:10.1074/jbc.m115.650176

Cited by 22 publications

(43 citation statements)

References 27 publications

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“…Recent singlemolecule FRET data indicate that both RNase R and its homolog, the Rrp44 subunit of the eukaryotic exosome (22,23), advance 3′-to-5′ along dsRNA in steps of approximately four base pairs through internal elastic coupling of their binding and catalytic sites (24). RNase R mutants that lack the nuclease activity required for processive motion nonetheless may separate hybridized strands of RNA (21,25), presumably through an entirely different, distributive mechanism similar to that described for DEAD-box helicases under certain conditions (26). Although RNase R appears to have a minor preference for adenine (13), no significant sequence-dependent behavior has been reported for this enzyme.…”

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Direct observation of processive exoribonuclease motion using optical tweezers

Fazal

Koslover

Luisi

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Bacterial RNases catalyze the turnover of RNA and are essential for gene expression and quality surveillance of transcripts. In Escherichia coli, the exoribonucleases RNase R and polynucleotide phosphorylase (PNPase) play critical roles in degrading RNA. Here, we developed an optical-trapping assay to monitor the translocation of individual enzymes along RNA-based substrates. Single-molecule records of motion reveal RNase R to be highly processive: one molecule can unwind over 500 bp of a structured substrate. However, enzyme progress is interrupted by pausing and stalling events that can slow degradation in a sequence-dependent fashion. We found that the distance traveled by PNPase through structured RNA is dependent on the A+U content of the substrate and that removal of its KH and S1 RNA-binding domains can reduce enzyme processivity without affecting the velocity. By a periodogram analysis of single-molecule records, we establish that PNPase takes discrete steps of six or seven nucleotides. These findings, in combination with previous structural and biochemical data, support an asymmetric inchworm mechanism for PNPase motion. The assay developed here for RNase R and PNPase is well suited to studies of other exonucleases and helicases.single-molecule biophysics | optical trap | exoribonuclease | processivity | step size B oth polynucleotide phosphorylase (PNPase) and RNase R are responsible for degrading a variety of transcripts in bacteria, such as mRNAs, defective rRNAs, and antisense regulatory RNAs (1-4). Although strains of Escherichia coli remain viable with a loss of either enzyme, the elimination of both results in an accumulation of rRNA fragments and cell death (3). To degrade RNA, both ribonucleases require an unstructured binding site of at least 7-10 nt at the 3′ end of the substrate (5-7), a sequence that often is supplied by posttranscriptional polyadenylation (8-10). After binding RNA, PNPase and RNase R processively digest the substrate in the 3′-to-5′ direction (11-13).PNPase consists of a trimer that degrades RNA at catalytic sites within a central channel (9,14). The enzyme is homologous to eukaryotic and archaeal exosomes and, like these hexameric proteins, possesses a core of six RNase PH domains arranged in a ringlike configuration (two per protomer) (6, 15). Each protomer also carries a KH and an S1 RNA-binding domain at its C terminus, both of which are connected to the body of the enzyme by flexible linkers (6, 16). These domains are thought to bind RNA upstream of the catalytic sites, and recent structural evidence suggests that they may guide the substrate into the central channel through a "hands gripping a rope" mechanism (16,17). By itself, PNPase may stall on structured RNA, particularly on G:C hairpins comprising more than six base pairs (18). PNPase often associates with the DEAD-box helicase RhlB to move through structured regions and generally does so in the context of the multienzyme degradosome (2, 19). However, there is some evidence that PNPase can digest structured tra...

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confidence: 99%

Direct observation of processive exoribonuclease motion using optical tweezers

Fazal

Koslover

Luisi

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…In the E. coli protein, we also identified Walker A and Walker B motifs that are responsible for ATP binding and consequent RNA helicase activity, and found that they are conserved in most mesophilic bacterial genera, but are absent from thermophilic bacteria (8), suggesting that the helicase is important for RNase R function. This conclusion was reinforced by biochemical analyses showing that the helicase activity is essential for efficient nuclease activity in vitro, particularly at lower temperatures, with duplexes containing short 3Ј overhangs, and with more stable RNA duplexes (8,9). Whether the helicase activity is also important for nuclease activity in vivo has been unclear.…”

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“…Escherichia coli RNase R contains an intrinsic RNA helicase activity (10), and recent work from our laboratory characterized this helicase and defined its relation to overall nuclease activity (8,9). In the E. coli protein, we also identified Walker A and Walker B motifs that are responsible for ATP binding and consequent RNA helicase activity, and found that they are conserved in most mesophilic bacterial genera, but are absent from thermophilic bacteria (8), suggesting that the helicase is important for RNase R function. This conclusion was reinforced by biochemical analyses showing that the helicase activity is essential for efficient nuclease activity in vitro, particularly at lower temperatures, with duplexes containing short 3Ј overhangs, and with more stable RNA duplexes (8,9).…”

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“…These enzymes play important roles in many aspects of RNA metabolism including mRNA and stable RNA turnover (2,3) and rRNA maturation (4). In contrast to most exoribonucleases, RNase R is particularly adept at degrading highly structured RNA molecules (2,(5)(6)(7), and defining the mechanism by which it carries out this process is highly important (8,9). Escherichia coli RNase R contains an intrinsic RNA helicase activity (10), and recent work from our laboratory characterized this helicase and defined its relation to overall nuclease activity (8,9).…”

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confidence: 99%

“…In contrast to most exoribonucleases, RNase R is particularly adept at degrading highly structured RNA molecules (2,(5)(6)(7), and defining the mechanism by which it carries out this process is highly important (8,9). Escherichia coli RNase R contains an intrinsic RNA helicase activity (10), and recent work from our laboratory characterized this helicase and defined its relation to overall nuclease activity (8,9). In the E. coli protein, we also identified Walker A and Walker B motifs that are responsible for ATP binding and consequent RNA helicase activity, and found that they are conserved in most mesophilic bacterial genera, but are absent from thermophilic bacteria (8), suggesting that the helicase is important for RNase R function.…”

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Helicase Activity Plays a Crucial Role for RNase R Function in Vivo and for RNA Metabolism

Hossain

Deutscher

2016

Journal of Biological Chemistry

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RNase R is a 3 to 5 hydrolytic exoribonuclease that has the unusual ability to digest highly structured RNA. The enzyme possesses an intrinsic, ATP-dependent RNA helicase activity that is essential in vitro for efficient nuclease activity against double-stranded RNA substrates, particularly at lower temperatures, with more stable RNA duplexes, and for duplexes with short 3 overhangs. Here, we inquired whether the helicase activity was also important for RNase R function in vivo and for RNA metabolism. We find that strains containing a helicase-deficient RNase R due to mutations in its ATP-binding Walker motifs exhibit growth defects at low temperatures. Most importantly, cells also lacking polynucleotide phosphorylase (PNPase), and dependent for growth on RNase R, grow extremely poorly at 34, 37, and 42°C and do not grow at all at 31°C. Northern analysis revealed that in these cells, fragments of 16S and 23S rRNA accumulate to high levels, leading to interference with ribosome maturation and ultimately to cell death. These findings indicate that the intrinsic helicase activity of RNase R is required for its proper functioning in vivo and for effective RNA metabolism.RNase R and its homologues are processive, 3Ј to 5Ј exoribonucleases present in organisms from bacteria to higher eukaryotes (1). These enzymes play important roles in many aspects of RNA metabolism including mRNA and stable RNA turnover (2, 3) and rRNA maturation (4). In contrast to most exoribonucleases, RNase R is particularly adept at degrading highly structured RNA molecules (2, 5-7), and defining the mechanism by which it carries out this process is highly important (8, 9). Escherichia coli RNase R contains an intrinsic RNA helicase activity (10), and recent work from our laboratory characterized this helicase and defined its relation to overall nuclease activity (8, 9). In the E. coli protein, we also identified Walker A and Walker B motifs that are responsible for ATP binding and consequent RNA helicase activity, and found that they are conserved in most mesophilic bacterial genera, but are absent from thermophilic bacteria (8), suggesting that the helicase is important for RNase R function. This conclusion was reinforced by biochemical analyses showing that the helicase activity is essential for efficient nuclease activity in vitro, particularly at lower temperatures, with duplexes containing short 3Ј overhangs, and with more stable RNA duplexes (8, 9). Whether the helicase activity is also important for nuclease activity in vivo has been unclear.It is known that RNase R can complement the essential function of CsdA, a DEAD-box RNA helicase, during cold shock (11), implying that the helicase activity of RNase R can function in vivo. Moreover, this complementation activity persists even when the nuclease activity of RNase R has been eliminated by mutation. However, all of these studies were carried out under conditions in which RNase R was overexpressed, raising the question of whether the helicase ever functions physiologically in vivo indep...

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