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Amy E Pasquinelli 《The EMBO journal》2012,31(19):3790-3791
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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
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3′-Azido-3′-Deoxythymidine (AZT) Mediates Cross-Resistance to Nucleoside Analogs in the Case of AZT-Resistant Human Immunodeficiency Virus Type 1 Variants 下载免费PDF全文
Eric J. Arts Miguel E. Quiones-Mateu Jamie L. Albright James-Paul Marois Charles Hough Zhengxian Gu Mark A. Wainberg 《Journal of virology》1998,72(6):4858-4865
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