Conserved Bacterial RNase YbeY
Plays Key Roles in 70S Ribosome
Quality Control and 16S rRNA Maturation
Asha Ivy Jacob,1Caroline Ko ¨hrer,1Bryan William Davies,2Uttam Lal RajBhandary,1and Graham Charles Walker1,*
1Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
2Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115, USA
Quality control of ribosomes is critical for cellular
function since protein mistranslation leads to severe
physiological consequences. We report evidence of
a previously unrecognized ribosome quality control
system in bacteria that operates at the level of 70S
to remove defective ribosomes. YbeY, a previously
unidentified endoribonuclease, and the exonuclease
RNase R act together by a process mediated specif-
ically by the 30S ribosomal subunit, to degrade
defective 70S ribosomes but not properly matured
70S ribosomes or individual subunits. Furthermore,
there is essentially no fully matured 16S rRNA in a
DybeY mutant at 45?C, making YbeY the only endor-
ibonuclease to be implicated in the critically impor-
tant processing of the 16S rRNA 30terminus. These
key roles in ribosome quality control and maturation
indicate why YbeY is a member of the minimal bacte-
rial gene set and suggest that it could be a potential
target for antibacterial drugs.
Given the intricacies of ribosomal RNA (rRNA) and ribosome
biogenesis, quality control mechanisms are critically required
to eliminate defective ribosomes and thus ensure proper protein
translation. Studies to date have led to the conclusion that ribo-
some quality control in bacteria acts mainly at the level of the
unassembled 30S and 50S subunits (Basturea et al., 2011;
Deutscher, 2009). Although a late-stage ribosome qualitycontrol
system (nonfunctional rRNA decay [NRD]) that can act at the
level of fully assembled ribosomes has been characterized in
eukaryotes (Cole et al., 2009; LaRiviere et al., 2006), a similar
system by which bacteria can specifically eliminate defective
70S ribosomes has not been reported (LaRiviere et al., 2006).
In this paper, we show that a highly conserved, previously
unidentified RNase, YbeY, plays critical roles in a hitherto unde-
scribed mechanism of late-stage 70S ribosome quality control in
YbeY (UPF0054 protein family) is found in nearly every
sequenced bacterium (Davies and Walker, 2008; Sonnhammer
et al., 1997). Also, ybeY is one of the 206 genes postulated to
comprise the minimal bacterial genome set (Gil et al., 2004).
ybeY is essential in some bacteria (Akerley et al., 2002; Kobaya-
shi et al., 2003), whereas in others (e.g., Escherichia coli and
Sinorhizobium meliloti), ybeY is not essential but its loss sensi-
tizes cells to a wide variety of physiologically diverse stresses,
including heat (Davies et al., 2010; Davies and Walker, 2008;
Rasouly et al., 2009). Structural studies of E. coli YbeY and its
dent hydrolase. Despite these structural insights and extensive
screening, the biochemical activity of YbeY and its orthologs
has remained elusive (Oganesyan et al., 2003; Penhoat et al.,
2005; Zhan et al., 2005).
Our recent studies of the physiological roles of YbeY led us
to consider the possibility that it might be an RNase, rather
than a protease as has been previously suggested (Oganesyan
et al., 2003; Penhoat et al., 2005; Zhan et al., 2005). We recently
found that YbeY is involved in the processing of all three rRNAs
(Davies et al., 2010). Furthermore, ybeY shows strong genetic
interactions with rnc, rnr, and pnp, whose products RNase III,
RNase R, and PNPase play important roles in both rRNA matu-
ration and RNA degradation (Bollenbach et al., 2005; Davies
et al., 2010; Deutscher, 2009; Purusharth et al., 2007; Walter
et al., 2002). The almost complete lack of properly matured
16S rRNA 30termini in the DybeY Drnr and DybeY Dpnp mutants
(Davies et al., 2010) was of particular interest because no
RNase has yet been implicated in this critically important pro-
cessing step (Deutscher, 2003). Additional observations that
tie YbeY to RNA metabolism include our finding that the
S. meliloti YbeY ortholog SMc01113 affects the regulation of
some sRNAs and their messenger RNA (mRNA) targets, and
the structural similarities that bacterial YbeYs share with the
MID domain of the eukaryotic Argonaute protein (Pandey
et al., 2011).
Here, we show that YbeY is a single strand specific endoribo-
nuclease that plays key roles in two crucial physiological func-
tions, a hitherto unrecognized late-stage 70S ribosome quality
control system that is particularly important under stress, and
in processing of the 16S rRNA 30terminus. These critical roles
of YbeY account for its presence in most bacterial genomes
and its inclusion in the minimal bacterial gene set.
Molecular Cell 49, 427–438, February 7, 2013 ª2013 Elsevier Inc. 427
YbeY Is a Metal-Dependent Ribonuclease
YbeY belongs to the UPF0054 family characterized by a three
histidine H3XH5XH motif that coordinates a metal ion thought
to be zinc. We report that purified YbeY is an RNase that
degrades total rRNA and mRNA effectively (Figures 1A and
1B). YbeY is substantially less effective at degrading total
tRNA in vitro (Figure 1A). For example, when a mixture of rRNA
and transfer RNA (tRNA) was used as a substrate, rRNA was
degraded but tRNA was not (Figure 1A). However, partial degra-
Figure 1. YbeY Is a Metal-Dependent Single
Purified YbeY was used at a concentration of 5 mM
in all assays unless mentioned otherwise.
(A) YbeY is able to degrade total rRNA (3.8 mM)
isolated from E. coli. tRNA (4.5 mM) is a relatively
poor substrate for YbeY. When rRNA (3.8 mM) and
tRNA (4.5 mM) were mixed, YbeY preferentially
degraded the rRNA. YbeY is inhibited by 50 mM
EDTA. RNase A (5 mM) was used as control to
show degradation of the mixture of rRNA (3.8 mM)
and tRNA (4.5 mM). The positions of the 23S, 16S,
5S rRNA, and tRNA are indicated.
(B) YbeY cleaves folA mRNA (4.0 mM) generated
by in vitro transcription. Digestion products were
analyzed by Synergel/agarose gel electropho-
(C–G) In vitro cleavage assay to identify the sub-
strate requirement of YbeY with short synthetic
with YbeY (5.0 mM) and 50
ibonucleotides (5.0 mM); ssRNA (C), dsRNA (D),
dsRNA containing a single-stranded extension at
the 30(E), hairpin substrate with perfectly base
paired blunt ends (F), and ssRNAs 7, 10, 12, and
20 nt long (G). Digestion products were analyzed
by polyacrylamide gel electrophoresis. OH–, alkali
ladder. ; indicates sites of cleavage by YbeY on
See also Figure S1 and S2.
dation of tRNA was observed when
YbeY was approximately in 2-fold excess
compared to the substrate (Figure S1A
available online). RNase A, used at the
same concentration as YbeY, efficiently
degraded both rRNA and tRNA (Fig-
ure 1A). As expected for an RNase,
YbeY was unable to degrade either
double or single-stranded DNA (Figures
activity, the RNase activity of YbeY was
inhibited by 50 mM EDTA (Figure 1A).
YbeY Is a Single Strand-Specific
To further characterize the RNase activity
of YbeY, we used a synthetic 30 nucleo-
tide (nt) RNA substrate that mimics the 30terminus of 16S
rRNA in its unprocessed form. It contains 18 nt of the mature
30terminus of 16S rRNA and 12 nt of the 30terminal precursor
sequence. YbeY can bind to this 30 nt single-stranded RNA
(ssRNA) in a concentration-dependent manner in a gel shift
assay (Figure S1D), albeit weakly. We attribute this weak binding
due the assay being carried out at 4?C and not 37?C at which
YbeY efficiently degrades RNA. To elucidate the substrate re-
quirement of YbeY, equimolar amounts of protein and synthetic
oligonucleotide substrates was used. YbeY cleaved the 30 nt
ssRNA substrate, producing a distinct pattern that indicates
YbeY Ribosome Quality Control and Maturation
428 Molecular Cell 49, 427–438, February 7, 2013 ª2013 Elsevier Inc.
isolated as described previously (Etchegaray and Inouye, 1999) with minor
modifications as described in the Supplemental Experimental Procedures.
45?C, and after blocking transcription with rifampicin at 37?C and 45?C at the
indicated time points, or from purified ribosome fractions with a QIAGEN
RNeasy Plus Mini Kit. 16S and 23S rRNAs were separated with Synergel/
agarose gel electrophoresis as described (Wachi et al., 1999). A detailed
description of the mapping of the 50and 30termini of 16S rRNA is provided
in the Supplemental Experimental Procedures.
Western Blot and Northern Blot Methods
Boththesetechniques weredoneasper standardprotocol (Brownetal.,2004;
Gallagher et al., 2008). Details are provided in the Supplemental Experimental
Supplemental Information includes Supplemental Experimental Procedures
and six figures and can be found with this article online at http://dx.doi.org/
We thank lab members for their help. This study was supported by grants from
National Institute of Health GM31030 and the Deshpande Center to G.C.W.,
GM17151 to U.L.R., and P30 ES002109 to the MIT Center for Environmental
Health Sciences. G.C.W. is an American Cancer Society Professor.
Received: June 5, 2012
Revised: October 18, 2012
Accepted: November 21, 2012
Published: December 27, 2012
J.J. (2002). A genome-scale analysis for identification of genes required for
growth or survival of Haemophilus influenzae. Proc. Natl. Acad. Sci. USA 99,
Arraiano, C.M., Andrade, J.M., Domingues, S., Guinote, I.B., Malecki, M.,
Matos, R.G., Moreira, R.N., Pobre, V., Reis, F.P., Saramago, M., et al.
(2010). The critical role of RNA processing and degradation in the control of
gene expression. FEMS Microbiol. Rev. 34, 883–923.
Awano, N., Rajagopal, V., Arbing, M., Patel, S., Hunt, J., Inouye, M., and
Phadtare, S. (2010). Escherichia coli RNase R has dual activities, helicase
and RNase. J. Bacteriol. 192, 1344–1352.
Basturea, G.N., Zundel, M.A., and Deutscher, M.P. (2011). Degradation of
ribosomal RNA during starvation: comparison to quality control during
steady-state growth and a role for RNase PH. RNA 17, 338–345.
Bollenbach, T.J., Lange, H., Gutierrez, R., Erhardt, M., Stern, D.B., and
Gagliardi, D. (2005). RNR1, a 30-50exoribonuclease belonging to the
RNR superfamily, catalyzes 30maturation of chloroplast ribosomal RNAs in
Arabidopsis thaliana. Nucleic Acids Res. 33, 2751–2763.
Brown, T., Mackey, K., and Du, T. (2004). Analysis of RNA by northern and slot
blot hybridization. Curr. Protoc. Mol. Biol. Chapter 4, Unit 4.9.
Cameron, V., and Uhlenbeck, O.C. (1977). 30-Phosphatase activity in T4 poly-
nucleotide kinase. Biochemistry 16, 5120–5126.
Campbell, T.L., and Brown, E.D. (2008). Genetic interaction screens with
ordered overexpression and deletion clone sets implicate the Escherichia
coli GTPase YjeQ in late ribosome biogenesis. J. Bacteriol. 190, 2537–2545.
Casadaban, M.J., and Cohen, S.N. (1979). Lactose genes fused to exogenous
promoters in one step using a Mu-lac bacteriophage: in vivo probe for tran-
scriptional control sequences. Proc. Natl. Acad. Sci. USA 76, 4530–4533.
Chen, C., and Deutscher, M.P. (2010). RNase R is a highly unstable protein
regulated by growth phase and stress. RNA 16, 667–672.
Cheng, Z.F., and Deutscher, M.P. (2002). Purification and characterization of
the Escherichia coli exoribonuclease RNase R. Comparison with RNase II.
J. Biol. Chem. 277, 21624–21629.
Cole, S.E., LaRiviere, F.J., Merrikh, C.N., and Moore, M.J. (2009). A conver-
gence of rRNA and mRNA quality control pathways revealed by mechanistic
analysis of nonfunctional rRNA decay. Mol. Cell 34, 440–450.
Condon, C., and Putzer, H. (2002). The phylogenetic distribution of bacterial
ribonucleases. Nucleic Acids Res. 30, 5339–5346.
Davies, B.W., and Walker, G.C. (2008). A highly conserved protein of unknown
function is required by Sinorhizobium meliloti for symbiosis and environmental
stress protection. J. Bacteriol. 190, 1118–1123.
Davies, B.W., Ko ¨hrer, C., Jacob, A.I., Simmons, L.A., Zhu, J., Aleman, L.M.,
Rajbhandary, U.L., and Walker, G.C. (2010). Role of Escherichia coli YbeY,
a highly conserved protein, in rRNA processing. Mol. Microbiol. 78, 506–518.
Deutscher, M.P. (2003). Degradation of stable RNA in bacteria. J. Biol. Chem.
Deutscher, M.P. (2009). Maturation and degradation of ribosomal RNA in
bacteria. Prog. Mol. Biol. Transl. Sci. 85, 369–391.
Etchegaray, J.P., and Inouye, M. (1999). Translational enhancement by an
element downstream of the initiation codon in Escherichia coli. J. Biol.
Chem. 274, 10079–10085.
Gallagher, S., Winston, S.E., Fuller, S.A., and Hurrell, J.G. (2008).
Immunoblotting and immunodetection. Curr. Protoc. Mol. Biol. Chapter 10,
Ghosh, S., and Deutscher, M.P. (1999). Oligoribonuclease is an essential
component of the mRNA decay pathway. Proc. Natl. Acad. Sci. USA 96,
Gil, R., Silva, F.J., Pereto ´, J., and Moya, A. (2004). Determination of the core of
a minimal bacterial gene set. Microbiol. Mol. Biol. Rev. 68, 518–537.
Inoue, K., Alsina, J., Chen, J., and Inouye, M. (2003). Suppression of defective
ribosome assembly in a rbfA deletion mutant by overexpression of Era, an
essential GTPase in Escherichia coli. Mol. Microbiol. 48, 1005–1016.
Inoue, K., Chen, J., Tan, Q., and Inouye, M. (2006). Era and RbfA have overlap-
ping function in ribosome biogenesis in Escherichia coli. J. Mol. Microbiol.
Biotechnol. 11, 41–52.
Kaberdina, A.C., Szaflarski, W., Nierhaus, K.H., and Moll, I. (2009). An unex-
pected type of ribosomes induced by kasugamycin:alookintoancestral times
of protein synthesis? Mol. Cell 33, 227–236.
Kazakov, A.E., Vassieva, O., Gelfand, M.S., Osterman, A., and Overbeek, R.
(2003). Bioinformatics classification and functional analysis of PhoH homo-
logs. In Silico Biol. (Gedrukt) 3, 3–15.
Kobayashi, K., Ehrlich, S.D., Albertini, A., Amati, G., Andersen, K.K., Arnaud,
M., Asai, K., Ashikaga, S., Aymerich, S., Bessieres, P., et al. (2003). Essential
Bacillus subtilis genes. Proc. Natl. Acad. Sci. USA 100, 4678–4683.
LaRiviere, F.J., Cole, S.E., Ferullo, D.J., and Moore, M.J. (2006). A late-acting
quality control process for mature eukaryotic rRNAs. Mol. Cell 24, 619–626.
Li, Z., and Deutscher, M.P. (1995). The tRNA processing enzyme RNase T is
essential for maturation of 5S RNA. Proc. Natl. Acad. Sci. USA 92, 6883–6886.
Li, Z., Pandit, S., and Deutscher, M.P. (1999a). Maturation of 23S ribosomal
RNA requires the exoribonuclease RNase T. RNA 5, 139–146.
Li, Z., Pandit, S., and Deutscher, M.P. (1999b). RNase G (CafA protein) and
RNase E are both required for the 50maturation of 16S ribosomal RNA.
EMBO J. 18, 2878–2885.
Liang, W., and Deutscher, M.P. (2010). A novel mechanism for ribonuclease
regulation: transfer-messenger RNA (tmRNA) and its associated protein
SmpB regulate the stability of RNase R. J. Biol. Chem. 285, 29054–29058.
Liang, W., and Deutscher, M.P. (2012). Post-translational modification of
RNase Ris regulated bystress-dependent reduction intheacetylatingenzyme
Pka (YfiQ). RNA 18, 37–41.
YbeY Ribosome Quality Control and Maturation
Molecular Cell 49, 427–438, February 7, 2013 ª2013 Elsevier Inc. 437
Liang, X.H., Liu, Q., and Fournier, M.J. (2009). Loss of rRNA modifications in
the decoding center of the ribosome impairs translation and strongly delays
pre-rRNA processing. RNA 15, 1716–1728.
Mangiarotti, G., Turco, E., Ponzetto, A., and Altruda, F. (1974). Precursor 16S
RNA in active 30S ribosomes. Nature 247, 147–148.
Muth, G.W., Ortoleva-Donnelly, L., and Strobel, S.A. (2000). A single adeno-
sine with a neutral pKa in the ribosomal peptidyl transferase center. Science
Oganesyan, V., Busso, D., Brandsen, J., Chen, S., Jancarik, J., Kim, R., and
Kim, S.H. (2003). Structure of the hypothetical protein AQ_1354 from
Aquifex aeolicus. Acta Crystallogr. D Biol. Crystallogr. 59, 1219–1223.
Pagliarini, D.J., Calvo, S.E., Chang, B., Sheth, S.A., Vafai, S.B., Ong, S.E.,
Walford, G.A., Sugiana, C., Boneh, A., Chen, W.K., et al. (2008). A mitochon-
drial protein compendium elucidates complex I disease biology. Cell 134,
Pandey, S.P., Minesinger, B.K., Kumar, J., and Walker, G.C. (2011). A highly
conserved protein of unknown function in Sinorhizobium meliloti affects
sRNA regulation similar to Hfq. Nucleic Acids Res. 39, 4691–4708.
Penhoat, C.H., Li, Z., Atreya, H.S., Kim, S., Yee, A., Xiao, R., Murray, D.,
Arrowsmith, C.H., and Szyperski, T. (2005). NMR solution structure of
Thermotoga maritima protein TM1509 reveals a Zn-metalloprotease-like
tertiary structure. J. Struct. Funct. Genomics 6, 51–62.
Powers, T., and Noller, H.F. (1990). Dominant lethal mutations in a conserved
loop in 16S rRNA. Proc. Natl. Acad. Sci. USA 87, 1042–1046.
Powers, T., and Noller, H.F. (1993). Allele-specific structure probing of
plasmid-derived 16S ribosomal RNA from Escherichia coli. Gene 123, 75–80.
Purusharth, R.I., Madhuri, B., and Ray, M.K. (2007). Exoribonuclease R in
Pseudomonas syringae is essential for growth at low temperature and plays
a novel role in the 30end processing of 16 and 5 S ribosomal RNA. J. Biol.
Chem. 282, 16267–16277.
Rasouly, A., Schonbrun, M., Shenhar, Y., and Ron, E.Z. (2009). YbeY, a heat
shock protein involved in translation in Escherichia coli. J. Bacteriol. 191,
is required for optimal activity of the 30S ribosomal subunit. J. Bacteriol. 192,
Roy-Chaudhuri, B., Kirthi, N., and Culver, G.M. (2010). Appropriate maturation
and folding of 16S rRNA during 30S subunit biogenesis are critical for transla-
tional fidelity. Proc. Natl. Acad. Sci. USA 107, 4567–4572.
Sambrook, J., and Russell, D.W. (2001). Molecular Cloning. A Laboratory
Manual (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory).
Sato, A., Kobayashi, G., Hayashi, H., Yoshida, H., Wada, A., Maeda, M.,
Hiraga, S., Takeyasu, K., and Wada, C. (2005). The GTP binding protein Obg
homolog ObgE is involved in ribosome maturation. Genes Cells 10, 393–408.
ribosomes. Nature 254, 34–38.
Siibak, T., Peil, L., Do ¨nho ¨fer, A., Tats, A., Remm, M., Wilson, D.N., Tenson, T.,
and Remme, J. (2011). Antibiotic-induced ribosomal assembly defects result
fromchanges inthesynthesisofribosomalproteins.Mol. Microbiol.80,54–67.
Sonnhammer, E.L., Eddy, S.R., and Durbin, R. (1997). Pfam: a comprehensive
database of protein domain families based on seed alignments. Proteins 28,
Thompson, J., Kim, D.F., O’Connor, M., Lieberman, K.R., Bayfield, M.A.,
Gregory, S.T., Green, R., Noller, H.F., and Dahlberg, A.E. (2001). Analysis of
mutations at residues A2451 and G2447 of 23S rRNA in the peptidyltransfer-
ase active site of the 50S ribosomal subunit. Proc. Natl. Acad. Sci. USA 98,
Tu, C., Zhou, X., Tropea, J.E., Austin, B.P., Waugh, D.S., Court, D.L., and Ji, X.
(2009). Structure of ERA in complex with the 30end of 16S rRNA: implications
for ribosome biogenesis. Proc. Natl. Acad. Sci. USA 106, 14843–14848.
Verstraeten, N., Fauvart, M., Verse ´es, W., and Michiels, J. (2011). The univer-
sally conserved prokaryotic GTPases. Microbiol. Mol. Biol. Rev. 75, 507–542.
Vesper, O., Amitai, S., Belitsky, M., Byrgazov, K., Kaberdina, A.C., Engelberg-
Kulka, H., and Moll, I. (2011). Selective translation of leaderless mRNAs
by specialized ribosomes generated by MazF in Escherichia coli. Cell 147,
Wachi, M., Umitsuki, G., Shimizu, M., Takada, A., and Nagai, K. (1999).
Escherichia coli cafA gene encodes a novel RNase, designated as RNase G,
involved in processing of the 50end of 16S rRNA. Biochem. Biophys. Res.
Commun. 259, 483–488.
Walter, M., Kilian, J., and Kudla, J. (2002). PNPase activity determines the effi-
ciency of mRNA 30-end processing, the degradation of tRNA and the extent of
polyadenylation in chloroplasts. EMBO J. 21, 6905–6914.
Wireman, J.W., and Sypherd, P.S. (1974). In vitro assembly of 30S ribosomal
particles from precursor 16S RNA of Escherichia coli. Nature 247, 552–554.
Xu, Z., O’Farrell, H.C., Rife, J.P., and Culver, G.M. (2008). A conserved rRNA
methyltransferase regulates ribosome biogenesis. Nat. Struct. Mol. Biol. 15,
Zhan, C., Fedorov, E.V., Shi, W., Ramagopal, U.A., Thirumuruhan, R.,
Manjasetty, B.A., Almo, S.C., Fiser, A., Chance, M.R., and Fedorov, A.A.
(2005). The ybeY protein from Escherichia coli is a metalloprotein. Acta
Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 61, 959–963.
YbeY Ribosome Quality Control and Maturation
438 Molecular Cell 49, 427–438, February 7, 2013 ª2013 Elsevier Inc.