共查询到20条相似文献,搜索用时 31 毫秒
1.
Sk Tofajjen Hossain Arun Malhotra Murray P. Deutscher 《The Journal of biological chemistry》2015,290(25):15697-15706
RNase R, which belongs to the RNB family of enzymes, is a 3′ to 5′ hydrolytic exoribonuclease able to digest highly structured RNA. It was previously reported that RNase R possesses an intrinsic helicase activity that is independent of its ribonuclease activity. However, the properties of this helicase activity and its relationship to the ribonuclease activity were not clear. Here, we show that helicase activity is dependent on ATP and have identified ATP-binding Walker A and Walker B motifs that are present in Escherichia coli RNase R and in 88% of mesophilic bacterial genera analyzed, but absent from thermophilic bacteria. We also show by mutational analysis that both of these motifs are required for helicase activity. Interestingly, the Walker A motif is located in the C-terminal region of RNase R, whereas the Walker B motif is in its N-terminal region implying that the two parts of the protein must come together to generate a functional ATP-binding site. Direct measurement of ATP binding confirmed that ATP binds only when double-stranded RNA is present. Detailed analysis of the helicase activity revealed that ATP hydrolysis is not required because both adenosine 5′-O-(thiotriphosphate) and adenosine 5′-(β,γ-imino)triphosphate can stimulate helicase activity, as can other nucleoside triphosphates. Although the nuclease activity of RNase R is not needed for its helicase activity, the helicase activity is important for effective nuclease activity against a dsRNA substrate, particularly at lower temperatures and with more stable duplexes. Moreover, competition experiments and mutational analysis revealed that the helicase activity utilizes the same catalytic channel as the nuclease activity. These findings indicate that the helicase activity plays an essential role in the catalytic efficiency of RNase R. 相似文献
2.
Yulia Redko Sylvie Aubert Anna Stachowicz Pascal Lenormand Abdelkader Namane Fabien Darfeuille Marie Thibonnier Hilde De Reuse 《Nucleic acids research》2013,41(1):288-301
Protein complexes directing messenger RNA (mRNA) degradation are present in all kingdoms of life. In Escherichia coli, mRNA degradation is performed by an RNA degradosome organized by the major ribonuclease RNase E. In bacteria lacking RNase E, the existence of a functional RNA degradosome is still an open question. Here, we report that in the bacterial pathogen Helicobacter pylori, RNA degradation is directed by a minimal RNA degradosome consisting of Hp-RNase J and the only DExD-box RNA helicase of H. pylori, RhpA. We show that the protein complex promotes faster degradation of double-stranded RNA in vitro in comparison with Hp-RNase J alone. The ATPase activity of RhpA is stimulated in the presence of Hp-RNase J, demonstrating that the catalytic capacity of both partners is enhanced upon interaction. Remarkably, both proteins are associated with translating ribosomes and not with individual 30S and 50S subunits. Moreover, Hp-RNase J is not recruited to ribosomes to perform rRNA maturation. Together, our findings imply that in H. pylori, the mRNA-degrading machinery is associated with the translation apparatus, a situation till now thought to be restricted to eukaryotes and archaea. 相似文献
3.
RNase R is a processive 3′-5′ exoribonuclease with a high degree of conservation in prokaryotes. Although some bacteria possess additional hydrolytic 3′-5′ exoribonucleases such as RNase II, RNase R was found to be the only predicted one in the facultative intracellular pathogen Legionella pneumophila. This provided a unique opportunity to study the role of RNase R in the absence of an additional RNase with similar enzymatic activity. We investigated the role of RNase R in the biology of Legionella pneumophila under various conditions and performed gene expression profiling using microarrays. At optimal growth temperature, the loss of RNase R had no major consequence on bacterial growth and had a moderate impact on normal gene regulation. However, at a lower temperature, the loss of RNase R had a significant impact on bacterial growth and resulted in the accumulation of structured RNA degradation products. Concurrently, gene regulation was affected and specifically resulted in an increased expression of the competence regulon. Loss of the exoribonuclease activity of RNase R was sufficient to induce competence development, a genetically programmed process normally triggered as a response to environmental stimuli. The temperature-dependent expression of competence genes in the rnr mutant was found to be independent of previously identified competence regulators in Legionella pneumophila. We suggest that a physiological role of RNase R is to eliminate structured RNA molecules that are stabilized by low temperature, which in turn may affect regulatory networks, compromising adaptation to cold and thus resulting in decreased viability. 相似文献
4.
Abudureyimu Abula Xiaona Li Xing Quan Tingting Yang Yue Liu Hangtian Guo Tinghan Li Xiaoyun Ji 《Nucleic acids research》2021,49(8):4738
RNA 2′-O-methylation is widely distributed and plays important roles in various cellular processes. Mycoplasma genitalium RNase R (MgR), a prokaryotic member of the RNase II/RNB family, is a 3′-5′ exoribonuclease and is particularly sensitive to RNA 2′-O-methylation. However, how RNase R interacts with various RNA species and exhibits remarkable sensitivity to substrate 2′-O-methyl modifications remains elusive. Here we report high-resolution crystal structures of MgR in apo form and in complex with various RNA substrates. The structural data together with extensive biochemical analysis quantitively illustrate MgR’s ribonuclease activity and significant sensitivity to RNA 2′-O-methylation. Comparison to its related homologs reveals an exquisite mechanism for the recognition and degradation of RNA substrates. Through structural and mutagenesis studies, we identified proline 277 to be responsible for the significant sensitivity of MgR to RNA 2′-O-methylation within the RNase II/RNB family. We also generated several MgR variants with modulated activities. Our work provides a mechanistic understanding of MgR activity that can be harnessed as a powerful RNA analytical tool that will open up a new venue for RNA 2′-O-methylations research in biological and clinical samples. 相似文献
5.
In Escherichia coli, the cold shock response is exerted upon a temperature change from 37°C to 15°C and is characterized by induction of several cold shock proteins, including polynucleotide phosphorylase (PNPase), during acclimation phase. In E. coli, PNPase is essential for growth at low temperatures; however, its exact role in this essential function has not been fully elucidated. PNPase is a 3′-to-5′ exoribonuclease and promotes the processive degradation of RNA. Our screening of an E. coli genomic library for an in vivo counterpart of PNPase that can compensate for its absence at low temperature revealed only one protein, another 3′-to-5′ exonuclease, RNase II. Here we show that the RNase PH domains 1 and 2 of PNPase are important for its cold shock function, suggesting that the RNase activity of PNPase is critical for its essential function at low temperature. We also show that its polymerization activity is dispensable in its cold shock function. Interestingly, the third 3′-to-5′ processing exoribonuclease, RNase R of E. coli, which is cold inducible, cannot complement the cold shock function of PNPase. We further show that this difference is due to the different targets of these enzymes and stabilization of some of the PNPase-sensitive mRNAs, like fis, in the Δpnp cells has consequences, such as accumulation of ribosomal subunits in the Δpnp cells, which may play a role in the cold sensitivity of this strain. 相似文献
6.
7.
The endoribonuclease RNase E is a key enzyme in RNA metabolism for many bacterial species. In Escherichia coli, RNase E contributes to the majority of RNA turnover and processing events, and the enzyme has been extensively characterized as the central component of the RNA degradosome assembly. A similar RNA degradosome assembly has been described in the α-proteobacterium Caulobacter crescentus, with the interacting partners of RNase E identified as the Kreb''s cycle enzyme aconitase, a DEAD-box RNA helicase RhlB and the exoribonuclease polynucleotide phosphorylase. Here we report that an additional degradosome component is the essential exoribonuclease RNase D, and its recognition site within RNase E is identified. We show that, unlike its E. coli counterpart, C. crescentus RhlB interacts directly with a segment of the N-terminal catalytic domain of RNase E. The crystal structure of a portion of C. crescentus RNase E encompassing the helicase-binding region is reported. This structure reveals that an inserted segment in the S1 domain adopts an α-helical conformation, despite being predicted to be natively unstructured. We discuss the implications of these findings for the organization and mechanisms of the RNA degradosome. 相似文献
8.
RNA polyadenylation and degradation in different Archaea; roles of the exosome and RNase R
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Polyadenylation is a process common to almost all organisms. In eukaryotes, stable poly(A)-tails, important for mRNA stability and translation initiation, are added to the 3′ ends of most mRNAs. Contrarily, polyadenylation can stimulate RNA degradation, a phenomenon witnessed in prokaryotes, organelles and recently, for nucleus-encoded RNA as well. Polyadenylation takes place in hyperthermophilic archaea and is mediated by the archaeal exosome, but no RNA polyadenylation was detected in halophiles. Here, we analyzed polyadenylation in the third archaea group, the methanogens, in which some members contain genes encoding the exosome but others lack these genes. Polyadenylation was found in the methanogen, Methanopyrus kandleri, containing the exosome genes, but not in members which lack these genes. To explore how RNA is degraded in the absence of the exosome and without polyadenylation, we searched for the exoribonuclease that is involved in this process. No homologous proteins for any other known exoribonuclease were detected in this group. However, the halophilic archaea contain a gene homologous to the exoribonuclease RNase R. This ribonuclease is not able to degrade structured RNA better than PNPase. RNase R, which appears to be the only exoribonucleases in Haloferax volcanii, was found to be essential for viability. 相似文献
9.
10.
Hyunjee Lee HyeokJin Cho Jooyoung Kim Sua Lee Jungmin Yoo Daeho Park Gwangrog Lee 《Nucleic acids research》2022,50(4):1801
RNase H is involved in fundamental cellular processes and is responsible for removing the short stretch of RNA from Okazaki fragments and the long stretch of RNA from R-loops. Defects in RNase H lead to embryo lethality in mice and Aicardi-Goutieres syndrome in humans, suggesting the importance of RNase H. To date, RNase H is known to be a non-sequence-specific endonuclease, but it is not known whether it performs other functions on the structural variants of RNA:DNA hybrids. Here, we used Escherichia coli RNase H as a model, and examined its catalytic mechanism and its substrate recognition modes, using single-molecule FRET. We discovered that RNase H acts as a processive exoribonuclease on the 3′ DNA overhang side but as a distributive non-sequence-specific endonuclease on the 5′ DNA overhang side of RNA:DNA hybrids or on blunt-ended hybrids. The high affinity of previously unidentified double-stranded (ds) and single-stranded (ss) DNA junctions flanking RNA:DNA hybrids may help RNase H find the hybrid substrates in long genomic DNA. Our study provides new insights into the multifunctionality of RNase H, elucidating unprecedented roles of junctions and ssDNA overhang on RNA:DNA hybrids. 相似文献
11.
Jan Abendroth Anja Ollodart Emma S. V. Andrews Peter J. Myler Bart L. Staker Thomas E. Edwards Vickery L. Arcus Christoph Grundner 《The Journal of biological chemistry》2014,289(4):2139-2147
Ribonucleases (RNases) maintain the cellular RNA pool by RNA processing and degradation. In many bacteria, including the human pathogen Mycobacterium tuberculosis (Mtb), the enzymes mediating several central RNA processing functions are still unknown. Here, we identify the hypothetical Mtb protein Rv2179c as a highly divergent exoribonuclease. Although the primary sequence of Rv2179c has no detectable similarity to any known RNase, the Rv2179c crystal structure reveals an RNase fold. Active site residues are equivalent to those in the DEDD family of RNases, and Rv2179c has close structural homology to Escherichia coli RNase T. Consistent with the DEDD fold, Rv2179c has exoribonuclease activity, cleaving the 3′ single-strand overhangs of duplex RNA. Functional orthologs of Rv2179c are prevalent in actinobacteria and found in bacteria as phylogenetically distant as proteobacteria. Thus, Rv2179c is the founding member of a new, large RNase family with hundreds of members across the bacterial kingdom. 相似文献
12.
Ribonucleases play an important role in RNA metabolism. Yet, they are also potentially destructive enzymes whose activity must be controlled. Here we describe a novel regulatory mechanism affecting RNase R, a 3′ to 5′ exoribonuclease able to act on essentially all RNAs including those with extensive secondary structure. Most RNase R is sequestered on ribosomes in growing cells where it is stable and participates in trans-translation. In contrast, the free form of the enzyme, which is deleterious to cells, is extremely unstable, turning over with a half-life of 2 min. RNase R binding to ribosomes is dependent on transfer-messenger RNA (tmRNA)-SmpB, nonstop mRNA, and the modified form of ribosomal protein S12. Degradation of the free form of RNase R also requires tmRNA-SmpB, but this process is independent of ribosomes, indicating two distinct roles for tmRNA-SmpB. Inhibition of RNase R binding to ribosomes leads to slower growth and a massive increase in RNA degradation. These studies indicate a previously unknown role for ribosomes in cellular homeostasis. 相似文献
13.
Tara Kashav Ramgopal Nitharwal S. Arif Abdulrehman Azat Gabdoulkhakov Wolfram Saenger Suman Kumar Dhar Samudrala Gourinath 《PloS one》2009,4(10)
Replication initiation is a crucial step in genome duplication and homohexameric DnaB helicase plays a central role in the replication initiation process by unwinding the duplex DNA and interacting with several other proteins during the process of replication. N-terminal domain of DnaB is critical for helicase activity and for DnaG primase interactions. We present here the crystal structure of the N-terminal domain (NTD) of H. pylori DnaB (HpDnaB) helicase at 2.2 Å resolution and compare the structural differences among helicases and correlate with the functional differences. The structural details of NTD suggest that the linker region between NTD and C-terminal helicase domain plays a vital role in accurate assembly of NTD dimers. The sequence analysis of the linker regions from several helicases reveals that they should form four helix bundles. We also report the characterization of H. pylori DnaG primase and study the helicase-primase interactions, where HpDnaG primase stimulates DNA unwinding activity of HpDnaB suggesting presence of helicase-primase cohort at the replication fork. The protein-protein interaction study of C-terminal domain of primase and different deletion constructs of helicase suggests that linker is essential for proper conformation of NTD to interact strongly with HpDnaG. The surface charge distribution on the primase binding surface of NTDs of various helicases suggests that DnaB-DnaG interaction and stability of the complex is most probably charge dependent. Structure of the linker and helicase-primase interactions indicate that HpDnaB differs greatly from E.coli DnaB despite both belong to gram negative bacteria. 相似文献
14.
Stéphane Skouloubris Kamel Djaout Isabelle Lamarre Jean-Christophe Lambry Karine Anger Julien Briffotaux Ursula Liebl Hilde de Reuse Hannu Myllykallio 《Open biology》2015,5(6)
ThyX is an essential thymidylate synthase that is mechanistically and structurally unrelated to the functionally analogous human enzyme, thus providing means for selective inhibition of bacterial growth. To identify novel compounds with anti-bacterial activity against the human pathogenic bacterium Helicobacter pylori, based on our earlier biochemical and structural analyses, we designed a series of eighteen 2-hydroxy-1,4-naphthoquinones (2-OH-1,4-NQs) that target HpThyX. Our lead-like molecules markedly inhibited the NADPH oxidation and 2′-deoxythymidine-5′-monophosphate-forming activities of HpThyX enzyme in vitro, with inhibitory constants in the low nanomolar range. The identification of non-cytotoxic and non-mitotoxic 2-OH-1,4-NQ inhibitors permitted testing their in vivo efficacy in a mouse model for H. pylori infections. Despite the widely assumed toxicity of naphthoquinones (NQs), we identified tight-binding ThyX inhibitors that were tolerated in mice and can be associated with a modest effect in reducing the number of colonizing bacteria. Our results thus provide proof-of-concept that targeting ThyX enzymes is a highly feasible strategy for the development of therapies against H. pylori and a high number of other ThyX-dependent pathogenic bacteria. We also demonstrate that chemical reactivity of NQs does not prevent their exploitation as anti-microbial compounds, particularly when mitotoxicity screening is used to prioritize these compounds for further experimentation. 相似文献
15.
Purusharth RI Klein F Sulthana S Jäger S Jagannadham MV Evguenieva-Hackenberg E Ray MK Klug G 《The Journal of biological chemistry》2005,280(15):14572-14578
Endoribonuclease E, a key enzyme involved in RNA decay and processing in bacteria, organizes a protein complex called degradosome. In Escherichia coli, Rhodobacter capsulatus, and Streptomyces coelicolor, RNase E interacts with the phosphate-dependent exoribonuclease polynucleotide phosphorylase, DEAD-box helicase(s), and additional factors in an RNA-degrading complex. To characterize the degradosome of the psychrotrophic bacterium Pseudomonas syringae Lz4W, RNase E was enriched by cation exchange chromatography and fractionation in a glycerol density gradient. Most surprisingly, the hydrolytic exoribonuclease RNase R was found to co-purify with RNase E. Co-immunoprecipitation and Ni(2+)-affinity pull-down experiments confirmed the specific interaction between RNase R and RNase E. Additionally, the DEAD-box helicase RhlE was identified as part of this protein complex. Fractions comprising the three proteins showed RNase E and RNase R activity and efficiently degraded a synthetic stem-loop containing RNA in the presence of ATP. The unexpected association of RNase R with RNase E and RhlE in an RNA-degrading complex indicates that the cold-adapted P. syringae has a degradosome of novel structure. The identification of RNase R instead of polynucleotide phosphorylase in this complex underlines the importance of the interaction between endo- and exoribonucleases for the bacterial RNA metabolism. The physical association of RNase E with an exoribonuclease and an RNA helicase apparently is a common theme in the composition of bacterial RNA-degrading complexes. 相似文献
16.
RNase BN, the Escherichia coli homolog of RNase Z, was previously shown to act as both a distributive exoribonuclease and an endoribonuclease on model RNA substrates and to be inhibited by the presence of a 3′-terminal CCA sequence. Here, we examined the mode of action of RNase BN on bacteriophage and bacterial tRNA precursors, particularly in light of a recent report suggesting that RNase BN removes CCA sequences (Takaku, H., and Nashimoto, M. (2008) Genes Cells 13, 1087–1097). We show that purified RNase BN can process both CCA-less and CCA-containing tRNA precursors. On CCA-less precursors, RNase BN cleaved endonucleolytically after the discriminator nucleotide to allow subsequent CCA addition. On CCA-containing precursors, RNase BN acted as either an exoribonuclease or endoribonuclease depending on the nature of the added divalent cation. Addition of Co2+ resulted in higher activity and predominantly exoribonucleolytic activity, whereas in the presence of Mg2+, RNase BN was primarily an endoribonuclease. In no case was any evidence obtained for removal of the CCA sequence. Certain tRNA precursors were extremely poor substrates under any conditions tested. These findings provide important information on the ability of RNase BN to process tRNA precursors and help explain the known physiological properties of this enzyme. In addition, they call into question the removal of CCA sequences by RNase BN. 相似文献
17.
Functional characterization of Helicobacter pylori DnaB helicase 总被引:1,自引:1,他引:0
Helicobacter pylori causes gastric ulcer diseases and gastric adenocarcinoma in humans. Not much is known regarding DNA replication in H.pylori that is important for cell survival. Here we report the cloning, expression and characterization of H.pylori DnaB (HpDnaB) helicase both in vitro and in vivo. Among the DnaB homologs, only Escherichia coli DnaB has been studied extensively. HpDnaB showed strong 5′ to 3′ helicase and ATPase activity. Interestingly, H.pylori does not have an obvious DnaC homolog which is essential for DnaB loading on the E.coli chromosomal DNA replication origin (oriC). However, HpDnaB can functionally complement the E.coli DnaB temperature-sensitive mutant at the non-permissive temperature, confirming that HpDnaB is a true replicative helicase. Escherichia coli DnaC co-eluted in the same fraction with HpDnaB following gel filtration analysis suggesting that these proteins might physically interact with each other. It is possible that a functional DnaC homolog is present in H.pylori. The complete characterization of H.pylori DnaB helicase will also help the comparative analysis of DnaB helicases among bacteria. 相似文献
18.
Syed Arif Abdul Rehman Vijay Verma Mohit Mazumder Suman K. Dhar S. Gourinath 《Journal of bacteriology》2013,195(12):2826-2838
To better understand the poor conservation of the helicase binding domain of primases (DnaGs) among the eubacteria, we determined the crystal structure of the Helicobacter pylori DnaG C-terminal domain (HpDnaG-CTD) at 1.78 Å. The structure has a globular subdomain connected to a helical hairpin. Structural comparison has revealed that globular subdomains, despite the variation in number of helices, have broadly similar arrangements across the species, whereas helical hairpins show different orientations. Further, to study the helicase-primase interaction in H. pylori, a complex was modeled using the HpDnaG-CTD and HpDnaB-NTD (helicase) crystal structures using the Bacillus stearothermophilus
BstDnaB-BstDnaG-CTD (helicase-primase) complex structure as a template. By using this model, a nonconserved critical residue Phe534 on helicase binding interface of DnaG-CTD was identified. Mutation guided by molecular dynamics, biophysical, and biochemical studies validated our model. We further concluded that species-specific helicase-primase interactions are influenced by electrostatic surface potentials apart from the critical hydrophobic surface residues. 相似文献
19.
Small cytoplasmic RNA (scRNA) of Bacillus subtilis is the RNA component of the signal recognition particle. scRNA is transcribed as a 354-nt precursor, which is processed to the mature 271-nt scRNA. Previous work demonstrated the involvement of the RNase III-like endoribonuclease, Bs-RNase III, in scRNA processing. Bs-RNase III was found to cleave precursor scRNA at two sites (the 5′ and 3′ cleavage sites) located on opposite sides of the stem of a large stem-loop structure, yielding a 275-nt RNA, which was then trimmed by a 3′ exoribonuclease to the mature scRNA. Here we show that Bs-RNase III cleaves primarily at the 5′ cleavage site and inefficiently at the 3′ site. RNase J1 is responsible for much of the cleavage that releases scRNA from downstream sequences. The subsequent exonucleolytic processing is carried out largely by RNase PH. 相似文献
20.
RNase R and RNase II are the two representatives from the RNR family of
processive, 3′ to 5′ exoribonucleases in Escherichia
coli. Although RNase II is specific for single-stranded RNA, RNase R
readily degrades through structured RNA. Furthermore, RNase R appears to be
the only known 3′ to 5′ exoribonuclease that is able to degrade
through double-stranded RNA without the aid of a helicase activity.
Consequently, its functional domains and mechanism of action are of great
interest. Using a series of truncated RNase R proteins we show that the
cold-shock and S1 domains contribute to substrate binding. The cold-shock
domains appear to play a role in substrate recruitment, whereas the S1 domain
is most likely required to position substrates for efficient catalysis. Most
importantly, the nuclease domain alone, devoid of the cold-shock and S1
domains, is sufficient for RNase R to bind and degrade structured RNAs.
Moreover, this is a unique property of the nuclease domain of RNase R because
this domain in RNase II stalls as it approaches a duplex. We also show that
the nuclease domain of RNase R binds RNA more tightly than the nuclease domain
of RNase II. This tighter binding may help to explain the difference in
catalytic properties between RNase R and RNase II.Ribonucleases (RNases) play important roles in RNA metabolism. They are
responsible for the maturation of stable RNA and the degradation of RNA
molecules that are defective or no longer required by the cell. Both
maturation and degradation are initiated by endoribonucleolytic cleavage(s)
and completed by the action of exoribonucleases
(1). In Escherichia
coli, three, relatively nonspecific, 3′ to 5′ processive
exoribonucleases are responsible for degradation of RNA: RNase II, RNase R,
and polynucleotide phosphorylase
(PNPase).3 RNase II
and PNPase appear to be primarily responsible for mRNA decay
(2), although their precise
functions may differ (3).
However, mRNAs containing extensive secondary structure, such as repetitive
extragenic palindromic sequences, are degraded by PNPase
(4,
5) or RNase R
(5). Likewise, degradation of
highly structured regions of rRNA
(6) and tRNA
(7),4
is carried out by PNPase and/or RNase R. These findings suggest that PNPase
and RNase R are the universal degraders of structured RNAs in vivo,
leaving RNase II to act on relatively unstructured RNAs.Whether or not an RNase acts upon a particular RNA appears to depend upon
the specificity of the RNase and the accessibility of the RNA to that RNase
(1). Purified RNase R readily
degrades both single- and double-stranded RNA molecules
(5,
8), and it is the only known
3′ to 5′ exoribonuclease able to degrade through double-stranded
RNA without the aid of helicase activity. To degrade RNA molecules containing
double-stranded regions, RNase R requires a 3′ single-stranded overhang
at least 5 nucleotides long to serve as a binding site from which degradation
can be initiated (5,
8,
9).5
How RNase R then proceeds through the RNA duplex is of great interest. An
important step toward elucidating the mechanism of action of RNase R is to
determine the contribution that each of its domains makes to substrate binding
and exoribonuclease activity.Despite differences in their physiological roles and intrinsic substrate
specificities, RNase R and RNase II both belong to the widely distributed RNR
family of exoribonucleases
(10–12).
RNR family members are all large multidomain proteins with processive 3′
to 5′ hydrolytic exoribonuclease activity that share a common linear
domain organization. RNase R contains two cold-shock domains (CSD1 and CSD2)
near its N terminus, a central nuclease, or RNB domain, an S1 domain near the
C terminus, and a low complexity, highly basic region at the C terminus
(Fig. 1A). The
nuclease domain contains four highly conserved sequence motifs
(10,
11). Motif I contains four
conserved aspartate residues that are thought to coordinate two divalent metal
ions that facilitate a two-metal ion mechanism similar to that of DEDD family
exoribonucleases and the proofreading domains of many polymerases
(13,
14). CSDs
(15–17)
and S1 domains (18,
19) are well known examples of
RNA-binding domains. Interestingly, there are reports that both of these
domains can act as nucleic acid chaperones and unwind RNA
(20–29),
providing a possible explanation for the ability of RNase R to degrade
structured RNAs. The role of the basic region at the C terminus of RNase R is
unknown, but it may act as an RNA-binding domain and/or a mediator of
protein-protein interactions.Open in a separate windowFIGURE 1.Linear domain organization of RNase R and RNase II proteins. The
CSDs are colored in cyan and blue for CSD1 and CSD2,
respectively, the nuclease domains are in green, the S1 domains are
red, and the low complexity, highly basic region, found in RNase R
only, is in magenta. A, RNase R. RNase R full-length is the
full-length wild-type RNase R protein. RNase RΔCSDs
lacks both CSD1 and CSD2. RNase RΔBasic is missing the
low complexity, highly basic region. RNase RΔS1 is
missing both the S1 domain and the low complexity, highly basic region.
RNase RΔCSDsΔS1 consists of the
nuclease domain alone. B, RNase II. RNase II full-length is
the full-length wild-type RNase II protein. RNase
IIΔCSDsΔS1 contains the nuclease domain
alone.Crystal structures of E. coli wild-type RNase II and a D209N
catalytic site mutant in complex with single-stranded RNA have recently been
solved (14,
30). In these structures the
two CSDs and the S1 domain come together to form an RNA-binding clamp that
directs RNA to the catalytic center at the base of a narrow, basic channel
within the nuclease domain
(14,
30). Only single-stranded RNA
can be accommodated by the RNA-binding clamp and the nuclease domain channel,
which explains the single strand specificity of RNase II. It is expected that
RNase R will adopt a similar structure.In this study, we determine the contribution that each of the domains of
RNase R makes to RNA-binding and exoribonuclease activity. We show that the
CSDs and the S1 domain are important for substrate binding, although their
roles differ. Of most interest, we show that the nuclease domain alone of
RNase R is sufficient to degrade through double-stranded RNA, whereas the
nuclease domain of RNase II is unable to carry out this reaction. The nuclease
domain of RNase R also binds RNA more tightly, which may explain the
difference in catalytic properties between RNase R and RNase II. 相似文献