Mode of action of RNase BN/RNase Z on tRNA precursors: RNase BN does not remove the CCA sequence from tRNA.

Abstract

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 Co(2+) resulted in higher activity and predominantly exoribonucleolytic activity, whereas in the presence of Mg(2+), 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.

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Mode of Action of RNase BN/RNase Z on tRNA Precursors
RNaseBNDOESNOTREMOVETHECCASEQUENCEFROMtRNA*
Receivedforpublication,May4,2010,andinrevisedform,May13,2010 Published,JBCPapersinPress,May19,2010,DOI10.1074/jbc.M110.141101
Tanmay Dutta and Murray P. Deutscher1
FromtheDepartmentofBiochemistryandMolecularBiology,UniversityofMiamiMillerSchoolofMedicine,Miami, Florida 33101
RNase BN, the Escherichia coli homolog of RNase Z, was pre-
viously shown to act as both a distributive exoribonuclease and
an endoribonuclease on model RNA substrates and to be inhib-
ited by the presence of a 3?-terminal CCA sequence. Here, we
examinedthemodeofactionofRNaseBNonbacteriophageand
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 dis-
criminator nucleotide to allow subsequent CCA addition. On
CCA-containingprecursors,RNaseBNactedaseitheranexori-
bonucleaseorendoribonucleasedependingonthenatureofthe
addeddivalentcation.AdditionofCo2?resultedinhigheractiv-
ity and predominantly exoribonucleolytic activity, whereas in
the presence of Mg2?, RNase BN was primarily an endoribo-
nuclease. 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
processtRNAprecursorsandhelpexplaintheknownphysiolog-
ical properties of this enzyme. In addition, they call into ques-
tion the removal of CCA sequences by RNase BN.
3?-Maturation of tRNA precursors differs among organisms
depending on whether or not the universal 3?-terminal CCA
sequence is encoded (1–6). In eukaryotic cells, this sequence is
absent from tRNA precursors, and the 3?-processing step is
catalyzed by the endoribonuclease, RNase Z or 3?-tRNase,
which cleaves at the discriminator base to allow subsequent
CCA addition by tRNA nucleotidyltransferase (3, 5, 7). In con-
trast, in some bacteria, such as Escherichia coli, the CCA
sequence is encoded, and maturation of the 3? terminus is car-
riedoutbyexoribonucleases(8,9),whereasinotherorganisms,
such as Bacillus subtilis, both types of 3?-processing are opera-
tive (3). Surprisingly, in E. coli, despite the fact that the CCA
sequence is present in all known tRNA precursors, an RNase Z
homolog, termed RNase BN, is present (10). As a consequence,
it has been of considerable interest to understand the function
in E. coli of this apparently unnecessary enzyme.
RNase BN was originally discovered as an enzyme required
for the maturation of those bacteriophage T4 precursor tRNAs
that lack a CCA sequence (11). Subsequently, RNase BN was
shown to also be able to process the CCA-encoded host E. coli
tRNA precursors in vivo (8, 9). However, of the five E. coli
RNases able to carry out the 3?-processing reaction, RNase BN
wastheleastefficient(12,13).Thus,undernormalgrowthcon-
ditions, RNase BN is unlikely to function in total tRNA matu-
ration, although it cannot be ruled out that the enzyme may
participate in 3?-maturation of some select tRNA species (8).
However, because E. coli mutants lacking RNase BN grow
essentially normally (14–17), either such tRNAs must be non-
essential,ortheir3?-maturationcanalsobecarriedoutbyother
RNases.
RNase BN was initially thought to be an exoribonuclease
based on its ability to remove a mononucleotide residue from
the3?terminusofsynthetictRNAprecursors(11).Thisconclu-
sion was strengthened by the subsequent finding that the
enzymeisalsoazincphosphodiesterase(10).However,studyof
RNase Z enzymes from various other organisms showed that
these enzymes are endoribonucleases (6, 18, 19), and the E. coli
enzyme was also found to display endoribonuclease activity on
certain tRNA precursors in vitro (14, 20). To help clarify this
situation, we recently carried out a detailed analysis of RNase
BN/RNase Z action on a variety of oligoribonucleotide sub-
strates and showed that the enzyme can act both as a distribu-
tive exoribonuclease and an endoribonuclease depending on
thenatureofthesubstrateandonits3?-terminalstructure(21).
Forexample,thepresenceofaCCAsequenceora3?-phospho-
ryl group was found to inhibit RNase BN (11, 21). On the other
hand,inarecentstudy(20),itwassuggestedthatRNaseBNcan
act on E. coli tRNA precursors and that it can actually remove
the CCA sequence. Hence, the mode of action of RNase BN on
tRNAprecursors,theeffectoftheCCAsequenceontheactivity
of the enzyme, and what determines the catalytic specificity
have been unclear.
Inthisstudy,weanalyzedtheactionofRNaseBNonavariety
of E. coli and phage T4 tRNA precursors. We show that RNase
BN can act on tRNA precursors containing or lacking a CCA
sequence and that it can do so as either an exoribonuclease or
an endoribonuclease. However, its mode of cleavage is very
dependent on the assay conditions, particularly with regard to
the identity of the metal ion present. When conditions more
closely resemble those thought to occur in vivo, RNase BN
functions primarily as an endoribonuclease, although these are
not necessarily the conditions under which RNase BN is most
active.Thus,RNaseBNspecificityandefficiencycanbegreatly
affected by assay conditions. Moreover, certain tRNA precur-
* Thisworkwassupported,inwholeorinpart,byNationalInstitutesofHealth
Grant GM16317.
1To whom correspondence should be addressed: Dept. of Biochemistry and
MolecularBiology,UniversityofMiamiMillerSchoolofMedicine,P. O.Box
016129, Miami, FL 33101. Tel.: 305-243-3150; Fax: 305-243-3955; E-mail:
mdeutsch@med.miami.edu.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 30, pp. 22874–22881, July 23, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc.Printed in the U.S.A.
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sors are extremely poor substrates for RNase BN under any
condition tested, which may explain why RNase BN is so poor
at total tRNA maturation in vivo. These findings provide
important information on the catalytic capabilities of RNase
BNontRNAprecursorsandhelpexplaintheknownphysiolog-
ical properties of this enzyme.
EXPERIMENTAL PROCEDURES
Materials—T4 polynucleotide kinase, DNase I, and RNase
A were purchased from New England Biolabs. Calf intestine
alkalinephosphataseandproteinaseKwereobtainedfromFer-
mentas. T4 RNA ligase, NucAwayTMspin columns, and the
MEGAshortscriptTMkit were purchased from Ambion, Inc.
The genomic DNA isolation kit was obtained from Promega
(Madison, WI). ExpressHyb hybridization solution was pur-
chased from Clontech. [?-32P]ATP, [?-32P]ATP, [5?-32P]pCp,2
and GeneScreen Plus hybridization transfer membrane were
obtained from PerkinElmer Life Sciences. The GenEluteTM
PCR clean-up kit was purchased from Sigma. The KOD Hot
Start DNA polymerase was obtained from Novagen. SequaGel
fordenaturingurea-polyacrylamidegelswasfromNationalDiag-
nostics. The HisTrap HP column was obtained from Amersham
Biosciences. All other chemicals were reagent grade. RNase BN
wasoverexpressedandpurifiedasdescribedpreviously(14,21).
The purity of the RNase BN preparation was determined by
silverstainingofanoverloadedSDS-polyacrylamidegel(2.5?g
of the purified protein). Only a single band at ?35 kDa was
observed; no minor contaminating proteins were detected.
Isolation of DNA from Phage T4 and E. coli—50 ?g of DNase
I and 100 ?g of RNase A were added to 750 ?l of phage T4
preparation (1011plaque-forming units/?l), and the mixture
was incubated at 37 °C for 10 min. 150 ?l of lysis buffer (0.4 M
EDTA, pH 8.0, 1% SDS, and 50 mM Tris-HCl pH 8.0) and 10 ?l
ofproteinaseK(10?g/?l)werethenadded,andincubationwas
continued at 65 °C for 30 min. The mixture was then vortexed
for 10 min with 750 ?l of phenol/chloroform/isoamyl alcohol
(24:24:1) and centrifuged at 15,000 ? g for 5 min. The aqueous
layer was re-extracted with 750 ?l of chloroform/isoamyl alco-
hol(24:1),andtheDNAintheaqueouslayerwasprecipitatedat
?20 °C for 2 h in the presence of 0.1 volume of 0.3 M sodium
acetate, pH 5.0, and 0.7 volume of isopropyl alcohol. DNA was
recovered by centrifugation at 20,000 ? g, and the DNA pellet
was washed with 70% ethanol, air-dried, and dissolved in 50 ?l
of DNase-free water.
E. coli DNA was purified using the genomic DNA isolation
kit (Promega, Madison, WI), according to the manufacturer’s
protocol.B. subtilisDNAwasobtainedfromDr.ChaitanyaJain
(University of Miami).
Synthesis of tRNA Precursors—Genomic DNAs, obtained
as described above, were used as the templates in PCRs using
KOD Hot Start DNA polymerase to prepare mature or pre-
tRNA genes. In all cases, the forward primer contained a T7
RNApolymerasepromotersequence.PCRproductswerepuri-
fied using the GenEluteTMPCR clean-up kit and were used as
the templates in in vitro transcription reactions for the synthe-
sis of tRNAs using the MEGAshortscriptTMtranscription kit.
Transcriptionconditionswereaccordingtothemanufacturer’s
protocol.Uponcompletion,thereactionmixturewasextracted
twice with phenol/chloroform/isoamyl alcohol (25:24:1), and
thetRNAproductswereprecipitatedat?20 °Cinthepresence
of0.1volumeof20%potassiumacetateand2.5volumesof95%
ethanol for 1 h. The samples were centrifuged at 20,000 ? g for
15 min at 4 °C, followed by washing with 70% ethanol. The
supernatant fraction was discarded, and the RNA pellet was
air-dried and dissolved in 50 ?l of diethyl pyrocarbonate-
treated water.
Northern Blot Analysis—tRNA samples digested with RNase
BN were resolved on a 6% denaturing polyacrylamide gel in
0.5? Tris borate/EDTA buffer and transferred to a nitrocellu-
losemembranebyhorizontaltransferfor1.5hat150mAusing
0.5?Trisborate/EDTAasthetransfersolution.DNAoligonu-
cleotideprobescomplementarytothe5?-endofthetRNAwere
32P-labeled at their 5?-ends with T4 polynucleotide kinase.
Probes were allowed to anneal to the transferred RNA by over-
nightincubationinExpressHybhybridizationsolution,andthe
detected bands were visualized by PhosphorImager analysis
(GE Healthcare).
3?-End Labeling of tRNA—tRNAs were labeled at their 3?-
ends with [5?-32P]pCp using T4 RNA ligase in the presence of
unlabeled ATP at 4 °C for 16 h. 20 ?l of the labeling reaction
mixturecontained10pmoloftRNA,12pmolof[5?-32P]pCp,10
units of T4 RNA ligase, and 1? T4 RNA ligase buffer (0.05 M
Tris-HCl,pH7.8,0.01 MMgCl2,0.01 Mdithiothreitol,and1mM
unlabeled ATP). Unincorporated [32P]pCp was removed using
a NucAwayTMspin column. The 3?-terminal phosphate was
removed by treatment with calf intestine alkaline phosphatase.
The 20-?l dephosphorylation reaction contained 5 pmol of
[3?-32P]pCp-labeled tRNA, 1 mM dithiothreitol, 1? calf intes-
tine alkaline phosphatase buffer, and 1 unit of calf intestine
alkaline phosphatase. The reaction mixture was incubated at
37 °Cfor30minandthenextractedtwicewiththesamevolume
of phenol/chloroform/isoamyl alcohol (25:24:1). tRNAs were
precipitated at ?20 °C for 1 h in the presence of 0.1 volume of
20% potassium acetate and 2.5 volumes of 95% ethanol. The
tRNApelletwascollectedbycentrifugationat20,000?gfor15
min, air-dried, and dissolved in diethyl pyrocarbonate-treated
water.
3?-tRNA Processing Assay—Each 30-?l reaction mixture con-
tainingeither20mMHEPES,pH6.5,200mMpotassiumacetate,
and0.2mMCoCl2or10mMTris-HCl,pH7.5,5mMMgCl2,200
mMpotassiumacetate,?0.05?MtRNAsubstrates,and0.14?M
purifiedRNaseBNwasincubatedat37 °C.Portionsweretaken
at the indicated times, and the reaction was terminated by the
addition of 2 volumes of gel loading buffer (95% formamide, 20
mM EDTA, 0.05% SDS, 0.025% bromphenol blue, and 0.025%
xylene cyanol). Reaction products were resolved on 20% dena-
turing 7.5 M urea-polyacrylamide gels and visualized using a
STORM 840 phosphorimaging device (GE Healthcare). Quan-
tification was carried out using ImageQuant (GE Healthcare).
AMP Incorporation Assay—Assays were carried out in 20-?l
reaction mixtures containing 20 mM HEPES, pH 6.5, 200 mM
potassium acetate, 0.2 mM CoCl2, 0.11 ?M T4 tRNAPro-Sersub-
strate, 0.14 ?M purified RNase BN, 1.0 ?g of purified tRNA
2The abbreviations used are: pCp, cytidine 3?,5?-bis(phosphate); nt,
nucleotide(s).
ModeofActionofRNaseBN
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nucleotidyltransferase, 1 mM unlabeled CTP, and 22 pmol of
[?-32P]ATP. Reaction mixtures were incubated at 37 °C, and
two samples were withdrawn at the indicated times. 150 ?l of
0.5% (w/v) yeast RNA and 200 ?l of 10% trichloroacetic acid
were added to one set of samples to stop the reaction. The
samples were placed in ice for 30 min and then centrifuged at
16,000 ? g for 15 min at 4 °C. Pellets were collected and dis-
solved in 50 ?l of diethyl pyrocarbonate-treated water, and the
radioactivity associated with the pellets was determined by liq-
uid scintillation counting using an LS 6500 multipurpose scin-
tillation counter (Beckman Coulter, Inc.).
RNA loading dye (2 volumes) was added to the other set of
samples to stop the reaction. The products were resolved on a
denaturing 7.5 M urea-polyacrylamide gel and visualized using
the STORM 840 phosphorimaging device.
RESULTS
TheactionofE. coliRNaseBN/RNaseZontRNAprecursors
hasremainedunclear.Intheearlieststudies,RNaseBNwasshown
to be required for maturation of certain phage T4 tRNA precur-
sors that lack an encoded CCA sequence (11, 22), and such an
activity is consistent with that of other RNase Z enzymes, which
generally cleave after the discriminator residue (6, 18). Interest-
ingly,RNaseBNalsocouldactontheE.colitRNAprecursorana-
logtRNA-CCA-Cninvitro(23)andonE.colitRNAprecursorsin
vivo to generate mature tRNAs (9, 12), indicating that it could
process CCA-containing precursors and that the CCA sequence
wasretained.Thesedataalsoareconsistentwithourrecentstudies
that showed that RNase BN can remove residues following a
CCA sequence but that it slows dramatically at a CCA
sequence in model RNA substrates (21). Hence, the recent
report of Takaku and Nashimoto (20) that RNase BN cleaves
mature and precursor E. coli tRNAs to remove the CCA
sequence was quite surprising. As a consequence, we have re-
examined the action of RNase BN on representative E. coli
tRNA precursors that contain a CCA sequence and also on a
phage T4 precursor and a B. subtilis precursor that lack the
CCA sequence to ascertain the products of RNase BN action
and to determine whether RNase BN functions as an exoribo-
nuclease or endoribonuclease on these substrates.
ActionofRNaseBNonaCCA-lesstRNAPrecursor—RNaseZ
enzymes from most organisms cleave tRNA precursors lacking
a CCA sequence in an endonucleolytic fashion right after the
discriminatornucleotidetogenerateasubstrateforsubsequent
CCA addition by tRNA nucleotidyltransferase (1–3, 5, 24). To
determine the mode of action of RNase BN on a tRNA precur-
sor of this type, we prepared by in vitro transcription the phage
T4 dimeric tRNAPro-Serprecursor (Fig. 1A), known to be a sub-
strate for 3?-processing by RNase BN in vivo (23, 25, 26). This
RNA was treated with purified RNase BN, and the products
were analyzed in detail.
As shown in Fig. 1B, treatment with RNase BN led to the
disappearance of the32P-labeled dimeric precursor with the
concomitant increase in a slightly shorter product that
migrated in the position of the dimer from which the 3?-termi-
nal UAA residues have been removed. To confirm that RNase
BN removes the 3 precursor-specific residues from the 3?-end
of tRNASer, the RNase BN cleavage reaction was repeated
with nonradioactive precursor in the presence of tRNA
nucleotidyltransferase, CTP, and [?-32P]ATP. If the com-
plete UAA sequencewereremovedbyRNaseBN,tRNAnucle-
otidyltransferase would add CC-32P-A following the discrimi-
nator nucleotide. That this occurs is shown in Fig. 1C. No
FIGURE 1. Processing of phage tRNAPro-Serprecursor by RNase BN.
A, shown is the structure of phage T4 tRNAPro-Serdimeric precursor. The dis-
criminatornucleotidesareshowninboldface.B,uniformly32P-labeledphage
T4 tRNAPro-Ser(25 nM) was used as the substrate. Digestion was carried out in
thepresenceofCo2?atpH6.5with0.14?MRNaseBNfortheindicatedtimes.
Products were analyzed by 6% denaturing PAGE. C, [32P]AMP incorporation
was determined as described under “Experimental Procedures” using unla-
beledphageT4tRNAPro-Serprecursor(0.11?M)asthesubstrate.Portionswere
withdrawn at the indicated time points. High molecular weight products
were precipitated by 10% trichloroacetic acid, and radioactivity associated
withtheprecipitatewascountedinascintillationcounter.f,tRNAPro-SerGUAA,
tRNA nucleotidyltransferase; F, tRNAPro-SerG, tRNA nucleotidyltransferase,
RNaseBN;Œ,tRNAPro-SerGUAA,tRNAnucleotidyltransferase,RNaseBN.DandE,
phage T4 tRNAPro-Serdimeric precursor (12 nM), labeled at its 3?-end with
[32P]pC as described under “Experimental Procedures,” was used as the sub-
strate. Digestion with RNase BN (0.04 ?M) was carried out in the presence of
either Co2?at pH 6.5 (D) or Mg2?at pH 7.5 (E). Quantitation of the data in D
andEisshownbelowthegels.DisappearanceofthetRNAprecursor(P)(f)
and appearance of the 4-nt product (E) are presented as the percentage
of the initial amount of precursor. All bands shorter than the full-length
tRNAPro-Serprecursor are incomplete transcripts that are labeled with
[32P]pCp.
ModeofActionofRNaseBN
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[32P]AMP was incorporated into the dimeric precursor in the
absenceofRNaseBN,buttherewasconsiderableincorporation
(0.46pmol)whenRNaseBNwaspresent.Thislevelofincorpo-
ration amounted to ?85% of the tRNA precursor added. As a
control, [32P]AMP incorporation into tRNAPro-Serlacking the
UAA residues was also measured. In this case, incorporation
continued for 30 min and did not yet reach a limit. The higher
level in the presence of RNase BN appears to be due to some
cleavage of the dimeric precursor by RNase BN to individual
tRNAProand tRNASer, resulting in additional 3? termini for
CCA addition. This was confirmed by gel electrophoresis, which
showedmonomer-sizedtRNAproducts(datanotshown).
Of most interest to understanding the mode of action of
RNaseBNisthequestionofwhethertheUAAresiduesatthe3?
terminus of the dimeric precursor are removed by a single
endonucleolytic cleavage or by exoribonucleolytic trimming
because RNase BN has the capacity for either activity (21). To
answer this question, [32P]pCp was added to the 3? terminus,
followed by removal of the 3?-phosphoryl group by phospha-
tase. The resulting labeled precursor was treated with RNase
BN, and the cleavage products were analyzed on a 20% dena-
turing acrylamide gel. Fig. 1D shows that the major product
produced was a tetranucleotide, with essentially no [32P]CMP
evident. These data demonstrate that RNase BN removes the 3
extra nucleotides in pre-tRNAPro-Serby endonucleolytic cleav-
age rather than by exonucleolytic trimming.
Action of RNase BN on a tRNA Precursor with a Long
3?-Trailer—It was recently suggested that E. coli RNase Z can-
notcleavepre-tRNAswith3?-trailersequences?6nt(20).This
observation is surprising considering that RNase BN/RNase Z
can function to mature all E. coli tRNA precursors in a strain
lackingothertRNA-processingexoribonucleases(8,12).Tore-
examine this point in more detail, we prepared32P-labeled
B. subtilis pre-tRNAThr. This RNA contains tRNAThrplus an
83-nt 3?-trailer and is known to be cleaved by the B. subtilis
RNase Z after the discriminator nucleotide (18). The precursor
was treated with purified RNase BN, and the data from this
experiment are presented in Fig. 2. RNase BN efficiently
cleavedthe156-ntprecursortogenerate,asthemajorproducts,
the73-ntCCA-lesstRNAThrandasecond,slightlylongerprod-
uct corresponding to the 83-nt 3?-trailer. Interestingly, RNase
BN also cleaved the precursor at a second position within the
3?-trailertogenerateaproduct?115ntinlengthandasmaller
fragment from the 3?-trailer. This ?115-nt product slowly dis-
appearedwithtime,presumablyduetoanadditionalRNaseBN
cleavage after the discriminator residue that also generated
tRNAThr. Nevertheless, these data clearly show that RNase BN
can endonucleolytically cleave even long 3?-trailer sequences
fromtRNAprecursors.Asmallamountof[32P]AMPalsoaccu-
mulated with increasing time of reaction due to the exoribo-
nuclease activity of RNase BN (11, 21), most likely resulting
fromsubsequentactiononsomeoftheproductfragmentsgen-
erated by the initial endonucleolytic cleavages.
Action of RNase BN on CCA-containing tRNA Precursors—
To determine the mode of action of RNase BN on CCA-con-
taining tRNA precursors and to examine the conclusion of
Takaku and Nashimoto (20) that RNase BN removes the CCA
sequence, we prepared several E. coli precursors by in vitro
transcriptionandtreatedthemwithpurifiedenzyme.Wechose
initially pre-tRNAPheV(Fig. 3A), a substrate studied in detail by
Takaku and Nashimoto (20). Two variants were prepared con-
taining either 3 or 6 nt following the CCA sequence. As shown
in the Northern analyses in Fig. 3 (B and C), RNase BN con-
verted each precursor to the size of mature tRNAPhe. No prod-
ucts shorter than the mature form were detectable. Moreover,
RNase BN did not remove any residues from mature tRNAPhe
(Fig. 3D). Thus, contrary to the results of Takaku and Nashi-
moto (20), under the assay conditions used, we found no evi-
dence for removal of the CCA sequence by RNase BN.
To ascertain the mode of removal of the extra 3?-residues on
pre-tRNAPhe, [32P]CMP was added to the 3? terminus of each
precursor using RNA ligase, [32P]pCp, and phosphatase, and
the products generated by RNase BN action were analyzed by
PAGE (Fig. 3E). Interestingly, with the pre-tRNA containing 6
extra 3?-residues, both [32P]CMP and a32P-labeled 7-mer oli-
gonucleotide were released as the major products at a ratio of
?3:1,respectively(Fig.3E).Asmallamountofdinucleotidewas
also noted, suggesting that cleavage may also occur within the
3?-trailer. From these data, we concluded that RNase BN can
removeextraresiduesfollowingtheCCAsequencebyeitheran
exonucleolytic or endonucleolytic mechanism. Both exonu-
cleolytic and endonucleolytic products were also obtained
whentheshorterpre-tRNAPhewasthesubstrate,andCMPwas
again the major product (data not shown). Thus, under the
assay conditions used (Co2?, pH 6.5), the exonucleolytic mode
of processing predominates. However, as shown below, the
mode of processing can be altered dramatically by a change in
assay conditions.
The second E. coli tRNA precursor examined was that of
tRNASelC. In previous work from our laboratory (12), it was
shown that this tRNA was matured even in the absence of the
major tRNA-processing exoribonucleases, suggesting that
another enzyme, such as RNase BN, might be involved. Pre-
tRNASelCwith 4 nt following the CCA sequence (Fig. 4A) was
FIGURE 2. Processing of B. subtilis tRNAThrprecursor. Uniformly32P-la-
beled tRNAThrprecursor (0.26 ?M) containing 83 extra nt at its 3?-end was
treatedwithRNaseBN(0.14?M)inthepresenceofCo2?atpH6.5.Theresult-
ing products, displayed on the right, were analyzed by 8% denaturing PAGE.
ModeofActionofRNaseBN
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synthesized and treated with purified RNase BN. The data in
Fig. 4B show, by Northern analysis, that RNase BN readily con-
verted the precursor to the size of the mature tRNA. Identical
results were obtained with [?-32P]-labeled precursor (data not
shown),confirmingtheNortherndata.Furthermore,therewas
no reduction in size upon treatment of mature tRNASelCwith
RNase BN (Fig. 4C). Thus, for tRNASelCas well, there is no
evidence for removal of the CCA sequence.
Analysis of the mode of 3?-maturation using precursor
labeled at its 3? terminus with [32P]CMP revealed that RNase
BN action was almost exclusively exonucleolytic, releasing
only CMP (Fig. 4D). However, when the 3?-phosphoryl
group from pCp addition was not removed, only the endo-
nucleolytic product, a 5-nt long oligomer, was detectable
(Fig. 4E), in keeping with the known inhibition of the exori-
bonuclease activity by a 3?-phosphoryl group (21). As with
tRNAPhe, assay conditions dramatically affected the mode of
3?-processing of pre-tRNASelC(see below).
A third E. coli pre-tRNA, that of tRNACysT, which contained
4 nt following the CCA sequence (Fig. 5A), was also analyzed.
Treatment of this precursor also generated a product that
migrated with mature tRNACys(Fig. 5B), again providing no
evidence for CCA removal. Determination of the mode of
removal of the extra nucleotides using [3?-32P]CMP-labeled
substrate, as described above, showed that CMP was the major
product, indicating exoribonuclease action (Fig. 5C). However,
in this case, a significant amount of dinucleotide was also gen-
erated by the endoribonuclease activity (Fig. 5C). Some anom-
alous internal cleavage was also observed (Fig. 5C). Assay con-
ditions also affected processing of this tRNA precursor, as
shown below.
EffectofAssayConditionsonModeofRNaseBNAction—Pre-
viousworkshowedthattheoptimalinvitroassayconditionsfor
RNase BN were at pH 6.5 in the presence of Co2?(11, 21), and
those were the conditions initially used in this study. However,
because such conditions are unlikely to be present in vivo, we
FIGURE 3. Maturation of tRNAPheVprecursor by RNase BN. A, shown is the
structure of the E. coli tRNAPheprecursor. The discriminator nucleotide is
showninboldface.B,thetRNAPheprecursor(0.05?M)with6extra3?-residues
after the CCA sequence was treated with RNase BN (0.14 ?M), and the cleav-
age products were analyzed by 6% denaturing PAGE, followed by Northern
blotting with a 5?-probe. C, the conditions were the same as described for B
exceptthattheprecursorhas3extra3?-nt.D,theconditionswerethesameas
described for B except that the substrate was mature (M) tRNAPhe. E and F,
tRNAPhelabeled with [32P]pC at its 3?-end (48 nM) was treated with RNase BN
(0.14 ?M) in the presence of either Co2?at pH 6.5 (E) or Mg2?at pH 7.5 (F). The
cleavageproductswereanalyzedby20%denaturingPAGE.Quantitationofthe
datafromexperimentssimilartothoseinEandFisshownbelowthegels.Disap-
pearance of the precursor (P) (f) and generation of CMP (Œ) and 7-nt oligomer
(E)productsarepresentedasapercentageoftheinitialamountofprecursor.
FIGURE 4. Maturation of tRNASelCprecursor by RNase BN. A, shown is the
structure of the E. coli tRNASelCprecursor. The discriminator nucleotide is
shown in boldface. B, the tRNASelCprecursor (0.04 ?M) with 4 extra residues
after the CCA sequence was treated with RNase BN (0.14 ?M), and the cleav-
age products were analyzed by 6% denaturing PAGE, followed by Northern
blotting with a 5?-probe. C, the conditions were the same as described for B
except that the substrate was mature (M) tRNASelC. D, tRNASelC(0.04 ?M)
labeled with [32P]pC at its 3?-end was treated with RNase BN (0.3 ?g) in the
presence of Co2?at pH 6.5. Cleavage products were analyzed by 20% dena-
turing PAGE. E, the conditions were the same as described for D except that
the tRNA was labeled with [3?-32P]pCp. F, the conditions were the same as
described for D except that reaction was carried out with Mg2?at pH 7.5.
QuantitationofthedatafromexperimentssimilartothoseinDandFisshown
below the relevant gels. Disappearance of the tRNA precursor (P) (f) and
generation of CMP (Œ) are 5-nt oligomer (E) products are presented as a
percentage of the initial amount of tRNA precursor.
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re-examined the mode of RNase BN action on the various pre-
tRNAs using pH 7.5 in the presence of Mg2?. For the CCA-less
precursors,tRNAPro-Ser(Fig.1,DandE)andtRNAThr(datanot
shown), there was essentially no difference in the products
generated or in their amounts. However, there were dra-
matic alterations in the products generated when the CCA-
containing precursors were tested under the two assay condi-
tions. Thus, for tRNAPhe(Fig. 3, E and F) and tRNASelC(Fig. 4,
D and F), changing the conditions essentially eliminated the
exoribonucleaseactivity.Basedonthequantitation,theendori-
bonuclease activity on tRNAPheremained at the same level,
whereas with tRNASelC, it increased appreciably; but for both
substrates, total processing activity decreased markedly due to
thelossofexoribonucleaseaction.Incontrast,withtRNACysas
the substrate, changing the assay conditions completely elimi-
nated both the exoribonuclease and endoribonuclease activi-
ties, and the precursor remained unprocessed (Fig. 5D). These
data demonstrate that assay conditions can have a profound
effectonRNaseBNactivityandonitsmechanismofaction,and
this effect can vary substantially depending on the RNA sub-
strate and whether or not a CCA sequence is present.
To define more carefully which aspect of the assay condi-
tions is responsible for the mode of action of the enzyme, pH
or metal ion, pre-tRNASelCwas treated with RNase BN using
multiple assay conditions. As shown in Fig. 6A, in the presence
of Co2?, the predominant product at both pH 6.5 and 7.5 was
CMP. In contrast, in the presence of Mg2?(Fig. 6B), the pre-
dominant product was the 5-nt oligomer at both pH values.
Thesedataclearlyindicatethatthemetalionisthedetermining
FIGURE 5. Maturation of tRNACysTprecursor by RNase BN. A, shown is the
structure of the E. coli tRNACysprecursor. The discriminator nucleotide is
showninboldface.B,uniformly32P-labeledpre-tRNACys(0.05?M)wasusedas
thesubstrate.DigestionwascarriedoutinthepresenceofCo2?atpH6.5with
0.14 ?M RNase BN for the indicated times. Products were analyzed by 6%
denaturingPAGE.M,maturetRNACys.CandD,tRNACys(0.05?M)labeledwith
[32P]pC at its 3?-end was treated with RNase BN (0.14 ?M) in the presence of
Co2?atpH6.5(C)orMg2?atpH7.5(D).Thecleavageproductswereanalyzed
by 20% denaturing PAGE. Quantitation of the data in C and D is presented
below the gels. Disappearance of the tRNA precursor (P) (f) and generation
of CMP (Œ) and endonucleolytic (E) products are presented as a percentage
of the initial amount of precursor.
FIGURE 6. Effect of assay conditions on RNase BN specificity. [3?-32P]pC-
labeled pre-tRNASelC(0.04 ?M) was treated with RNase BN (0.14 ?g) in the
presence of Co2?at pH 6.5 or pH 7.5 (A) or in the presence of Mg2?at pH 6.5
or pH 7.5 (B). Cleavage products were analyzed by 20% denaturing PAGE.
ModeofActionofRNaseBN
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factorinwhetherRNaseBNfunctionsasanexoribonucleaseor
endoribonuclease.
DISCUSSION
The results of this study provide important information on
E. coli RNase BN/RNase Z catalysis and function. We have
shown that (a) RNase BN can process the 3? termini of both
CCA-less and CCA-containing pre-tRNAs; (b) contrary to the
suggestion of Takaku and Nashimoto (20), RNase BN does not
normally remove the CCA sequence from precursor or mature
tRNAs; (c) RNase BN can act as either an exoribonuclease or
endoribonuclease on tRNA precursors; and (d) the mode of
actionofRNaseBNisdependentonthemetalionpresent.This
combination of catalytic properties sets RNase BN apart from
otherRNaseZenzymesandraisesagainthequestionofwhatits
function might be in E. coli. Despite its ability to act on tRNA
precursors, there is no evidence that it does so in vivo except
when all the other 3?-processing enzymes are absent or during
bacteriophage T4 infection when it is required for T4 tRNA
maturation (22). In fact, cells lacking RNase BN display no dis-
cernible phenotype (16).
TheabilityofRNaseBNtoprocessbothCCA-lessandCCA-
containing tRNAs combines in this enzyme the catalytic prop-
ertiesofmostRNaseZenzymes,whichcleaveafterthediscrim-
inator nucleotide (11, 14), with those of the RNase Z from
Thermotoga maritima, which cleaves right after the CCA
sequence (6). This raises the question of why our results differ
from those of Takaku and Nashimoto (20). Those workers sug-
gested that E. coli RNase Z cleaved both mature tRNAs and
tRNA precursors to remove the 3?-terminal CCA sequence.
Aside from the fact that such a reaction would appear to be
wasteful,requiringcontinuedre-additionoftheCCAsequence,
we showed previously with model RNA substrates that RNase
BN action is inhibited by the CCA sequence except for minor
trimming of the terminal AMP residue (21). It is also known
that end turnover of tRNA in vivo is due to the action of RNase
T, not RNase BN (27). One possibility that might explain the
difference in results is that Takaku and Nashimoto carried out
all their studies at 50 °C with a much higher ratio of enzyme to
substrate (20). Perhaps, at that elevated temperature, tRNA
structurewouldbeatleastpartiallydisrupted,alteringthespec-
ificity of RNase BN for its substrates. However, when we tested
RNase BN activity on tRNAPheand tRNACysunder the condi-
tions used in Ref. 20, we were still unable to observe removal of
the CCA sequence.3Thus, further work will be necessary to
reconcile the differences in data between the two laboratories.
In previous work using model RNA substrates, we showed
that RNase BN had both exoribonuclease and endoribo-
nuclease activities (21). The studies presented here extend
those observations to tRNA precursors as well. With CCA-
less substrates, the mode of cleavage was almost exclusively
endonucleolytic, resulting in cleavage after the discrimina-
tor nucleotide; however, with CCA-containing tRNA pre-
cursors, either mode of action was possible and depended
primarily on the metal ion present. In the presence of Co2?,
RNase BN displayed increased activity and functioned
largely as an exoribonuclease to remove extra 3?-residues
from tRNA precursors. In the presence of Mg2?, its primary
mode of action was as an endoribonuclease, cleaving after
the CCA sequence. The role of the metal ion in the activity
and specificity of RNase BN is of considerable interest for
future structural and functional studies.
As noted, the action of RNase BN also varied dramatically
with the pre-tRNA substrate. Thus, CCA-less tRNAs were
cleaved endonucleolytically after the discriminator residue,
whereas with CCA-containing molecules, residues following
the CCA sequence could be removed either exonucleolytically
or endonucleolytically. How the enzyme distinguishes the two
types of substrates and what structural features of the tRNA
determine this specificity remain to be explored. It should also
benotedthatseveralpre-tRNAstestedwerepoorsubstratesfor
RNase BN. These included tRNACys, which was active with
Co2?but was essentially inactive in the presence of Mg2?(Fig.
5D). Pre-tRNALeuZand pre-tRNAAlaUalso were poorly acted
uponbyRNaseBNeveninthepresenceofCo2?.3Theinability
of RNase BN to function efficiently on all pre-tRNAs likely
explains why cells grow very slowly in the absence of all the
other 3?-tRNA-processing enzymes present in E. coli (12).
The results presented here add considerably to our knowl-
edgeofRNaseBNspecificityandhelpexplainwhatisknownat
present about its in vivo action on tRNA precursors. However,
there is clearly still much to be learned about this interesting
RNase with regard to its mechanism and its primary in vivo
function.
Acknowledgments—We thank Drs. Georgeta Basturea and Pavana-
puresan Vaidyanathan for helpful discussions and comments. We
also thank Drs. Georgeta Basturea, Chaitanya Jain, and Arun Mal-
hotra and Christie Taylor for reading and commenting on the manu-
script. We thank Dr. Chaitanya Jain for providing B. subtilis DNA.
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