Molecular paleontology: a biochemical model of the ancestral

13 Pages·2013·5.95 MB·English

Published online 25 January 2013 Nucleic Acids Research, 2013, Vol. 41, No. 5 3373–3385
doi:10.1093/nar/gkt023
Molecular paleontology: a biochemical model of the
ancestral ribosome
Chiaolong Hsiao1,2,3, Timothy K. Lenz1,2,3, Jessica K. Peters1,2,3, Po-Yu Fang1,2,3,
Dana M. Schneider1,2,3, Eric J. Anderson1,2,3, Thanawadee Preeprem3,4,
Jessica C. Bowman1,2,3, Eric B. O’Neill1,2,3, Lively Lie2,3,4, Shreyas S. Athavale2,3,4,
J. Jared Gossett2,3,4, Catherine Trippe1,2,3, Jason Murray1,2,3, Anton S. Petrov1,2,3,4,
Roger M. Wartell2,3,4, Stephen C. Harvey1,2,3,4, Nicholas V. Hud1,2,3 and
Loren Dean Williams1,2,3,* Do
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1School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332-0400, USA, ad
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2Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA d
30332-0363, USA, 3Center for Ribosomal Origins and Evolution, Georgia Institute of Technology, Atlanta, GA fro
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30332-0400, USA and 4School of Biology, Georgia Institute of Technology, Atlanta, GA 30332-0230, USA h
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Received September 5, 2012; Revised December 7, 2012; Accepted January 3, 2013 ://a
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ABSTRACT catalyzes formation of peptide bonds. Some components .o
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of the ribosome are highly conserved throughout extant p
Ancient components of the ribosome, inferred from .c
life (1) and are considered to be among the oldest struc- o
m
a consensus of previous work, were constructed
tures in biology (2–7). The peptidyl transferase center /n
in silico, in vitro and in vivo. The resulting model of (PTC), for example, is thought to predate coded protein ar/a
theancestralribosomepresentedhereincorporates (8–12). If so, the PTC emerged from an ancient biolo- rtic
(cid:2)20% of the extant 23S rRNA and fragments of five gical world, possibly an ‘RNA World’ (13–17), before le
-a
ribosomal proteins. We test hypotheses thatances- life adopted all the processes of Crick’s ‘central dogma’ b
s
tral rRNA can: (i) assume canonical 23S rRNA-like (18). In this scenario, the PTC was an active participant tra
c
secondary structure, (ii) assume canonical tertiary in the origins of current biology and is one of our t/4
structure and (iii) form native complexes with ribo- most direct biochemical links to the distant evolutionary 1/5
past. /3
somal protein fragments. Footprinting experiments 3
support formation of predicted secondary and berSgev(e8r)a,lWgriolluiapms,sin(9c)luadnindgGFuotxel(l1a1n),dNHoallrevrey(12(1),),Shteaivne- 73/24
tertiary structure. Gel shift, spectroscopic and 1
proposed molecular-level events in early ribosomal evolu- 4
yeast three-hybrid assays show specific inter- tion or have determined universally conserved ribosomal 90
7
actions between ancestral rRNA and ribosomal components (Figure 1). These proposed evolutionary b
y
protein fragments, independent of other, more pathways can be used to predict specific sequences and g
u
recent, components of the ribosome. This robust- structures of ancestral rRNAs and polypeptides. Here, es
ness suggests that the catalytic core of the we use a consensus of proposed evolutionary pathways t on
ribosome is an ancient construct that has survived and conserved ribosomal components (Figure 1) to 05
billions ofyears of evolution without major changes design and construct a molecular-level model, in silico, Ap
in structure. Collectively, the data here support a in vitro and in vivo, of an ancestral PTC (a-PTC, ril 2
Figure 2). This model is intended as a starting platform 0
model in which ancestors of the large and small 1
for an iterative hypothesis-testing approach for under- 9
subunits originated and evolved independently of
standing the origins of translation. Our design process
each other, with autonomous functionalities.
relies substantially on three-dimensional (3D) structures,
whicharemoreconserved thansequence overlongevolu-
tionary time frames (9,19,20). The a-PTC incorporates
INTRODUCTION
fragments of the 23S rRNA, fragments of ribosomal
The ribosome is a molecular machine that synthesizes all proteins and divalent cations.
coded proteins. It is made of a small subunit (SSU) that The ancestral rRNA fragments inferred here from con-
decodes messenger RNA and a large subunit (LSU) that sensus(Figure1)arejoinedtogethertoformasingleRNA
*To whom correspondence should be addressed. Tel:[phone]; Fax:[phone]; Email: [email protected]
(cid:2)TheAuthor(s)2013.PublishedbyOxfordUniversityPress.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionNon-CommercialLicense(http://creativecommons.org/licenses/
by-nc/3.0/),whichpermitsunrestrictednon-commercialuse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
3374 NucleicAcidsResearch,2013,Vol.41,No.5
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Figure 1. Various models of 23S rRNA evolution. The dashed line illustrates the canonical secondary structure of the T. thermophilus 23S rRNA. 1/5
Secondarystructuraldomainsareindicatedbyromannumerals.TheredandgreenlinesshowthetwoinnershellsoftheribosomalonionofHsiao /3
and Williams, marking the rRNA that is in closest proximity, in three dimensions, to the site of peptidyl transfer. The gray boxes are ancient 37
aFcocxo.rdMinugltitdoenthtaet‘eAM-mgi2n+o-rp’hmosepthhaotdeoinftSerteaicntbioenrgs,.aTlhsoehparoshpeodsebdoaxsesa(nwiinthdibcalatcokr ohforainzocinetnatlrliRneNs)Aa,raereanicnideinctataecdcobrydimngagtoentthaecnirectlweso.rTkihnegoarnaanlgyesislinoef 3/24
1
showstheuniversallyconservedportionsofthe23SrRNAinbacteria,archaea,eukarya,andinmitochondria,asdeterminedbyGutellandHarvey. 4
9
0
7
polymer (a-rRNA, Figure 2). The a-rRNA contains inversely with radial distance from the site of peptidyl b
y
rRNA that forms and surrounds the PTC, which is transfer (9). g
u
composed of fragments from Domains II, IV and V. Ribosomal proteins do not engage directly in catalytic es
Using the 3D structure of the Thermus thermophilus processes in the ribosome (23,24). However, the non- t o
n
LSU (21), the 23S rRNA was ‘shaved’ to a rough sphere globular ‘tails’ of some ribosomal proteins do penetrate 0
5
of around 30A˚ in radius, centered at the site of peptidyl the LSU and interact extensively with the rRNA that A
p
tmraennstsfe.rT. oThfaiscilsihtaatveinrgecopnronceecstsioncreoaftetdhe1se3 frrRagNmAentfsratgo- fLo2r,mLs3t,hLe4c,aLta1ly5tiacnPdTLC2.2Tpheenettarialsteotfhreib3o0sAo˚msaplhperreottehinast ril 20
1
form a polymer, termini were selected preferentially in definesthea-rRNAandarehighlyconservedinconform- 9
A-form helical regions of the rRNA 3D structure and ation throughout the three domains of life (25–27). We
were capped with stem loops. The result is a single RNA shaved those proteins at the 30A˚ boundary used for the
polymer containing the most ancient 20% of the T. 23S rRNA, and included the resulting peptides in the
thermophilus 23S rRNA. The fragments are stitched a-PTC. The shaving process did not disrupt protein sec-
togetherbystemloopsinsuchawaythatthe3Dstructure ondary structure; none of these protein tails are globular.
of the PTC will be maintained (Figure 2). The a-rRNA Theancestralpeptidecomponentsofthea-PTCarecalled
containsrRNAthatis(i)universallyconservedinsecond- a-rPeptide L2, a-rPeptide L3, a-rPeptide L4, a-rPeptide
ary and 3D structure in extant organisms and organelles L15 and a-rPeptide L22 (see Table 1 for peptide se-
(1,9),(ii)tightlynetworkedbymolecularinteractions(11), quences). It was previously proposed that these tails are
(iii)denselycoordinatedbyMg2+ions(22)and(iv)found moreancientthananyproteinwithglobularstructure(9).
in the central shells of the ribosomal ‘onion’. The riboso- Using computation, in vitro and yeast three-hybrid ex-
mal onion is a conceptual model in which the age of periments,wetestthehypothesesthat:(i)a-rRNAadopts
elements of the ribosome is considered to correlate LSU-like secondary structure, as shown in Figure 2A,
NucleicAcidsResearch,2013,Vol.41,No.5 3375
Table 1. a-rPeptides derived from T. thermophilus ribosomal proteins
Parent rProtein Length a-rPeptide sequence
rProtein residue
range
L2 219–241 23 PHVRGAAMNPVDHPHGGGEGRAP
(a-rPeptide L2)
L3 136–153 18 RHPGSIGNRKTPGRVYKG
(a-rPeptide L3)
L4 52–92 41 KTRGEVAYSGRKIWPQKHTGRAR
HGDIGAPIFVGGGVVFGP
(a-rPeptide L4)
L15 19–63 45 VGRGPGSGHGKTATRGHKGQKSRS
GGLKDPRRFEGGRSTTLMRLP D
(a-rPeptide L15) o
w
L22 82–99 18 LKRVLPRARGRADIIKKR n
(a-rPeptide L22) loa
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the in silico model of the a-PTC. This a-PTC model m
h
(Figure 2B) contains 615 nucleotides of RNA, 505 of ttp
wofhwichhicahrearferofmromthe11Ta.dtdheedrmsotepmhilluoso2p3sSofrR10NnAucalneodti1d1e0s s://ac
a
each.Thea-PTCcontainsfivepeptidesderivedfromribo- d
e
somal proteins (Table 1). m
ic
The T. thermophilus 23S rRNA was shaved to yield the .o
u
rRNAfragmentsindicatedbytheblacklinesinFigure2A. p
.c
Theshavingprocessleft13rRNAfragments.Mostofthe o
m
fragmentterminiarewithinhelicalregions,allowingfacile /n
a
reconnectionwithstemloopstocreateacontinuousRNA r/a
chain. For 11 of the connections, the stem loop is 50- rtic
gccGUAAggc-30, which is a GNRA tetraloop (capital le
-a
letters) on a three base pair stem (lower case letters). b
s
Figure 2. (A)Predictedsecondarystructureoftheancestral23SrRNA. The stem loops, indicated by blue lines in Figure 2A, tra
Astrnuccetsutrrae,lfarraegmdeernivtsedoffrroRmNAa,coinndsiecnastueds obfymbloadckelslinoefsriRnNthAeseevcooluntdiaorny. were computationally docked onto the 3D a-rRNA ct/41
The ancestral rRNA elements are stitched together by stem loops model as described in Supplementary Figure S1. Two of /5
(blue). The RNA sequences are from the T. thermophilus 23S rRNA. the rRNA fragments were connected directly, without a /33
Helix numbers are indicated. The predicted secondary structure of the stem loop. 73
a-rRNAaloneishighlightedintheoutbox.(B)3Dmodelofthea-PTC. /2
4
This 3D model contains the a-rRNA plus five a-rPeptides (ancestral Stem-loop geometry 14
fragmentsofribosomalproteinsL2,L3,L4,L15andL22).a-rRNAis 9
Wehavepreviouslydescribedtheconsensusconformation 0
showninribbon(brown),thestemloopsareblueandthepeptidesare 7
in surface representation (green). For reference, A-site (yellow) and of a GNRA tetraloop (28). Here, we searched the crystal by
P-site(red)substratesareshowninthefigure,butarenotcomponents structure of the T. thermophilus ribosome (PDB entries g
u
ofthea-PTC.ThemodernLSUsurfaceisshownforcomparison(light 2J00 and 2J01) for a 10-nucleotide GUAA tetraloop es
gray, transparent). (50-gccGUAAggc-30) containing several additional base t o
n
pairs on the stem. An rRNA sub-structure from the 0
(ii) a-rRNA in association with Mg2+ adopts LSU-like SSU was selected based on the highest sequence identity 5 A
t(eiiri)tiaary-rRinNteAracftoiornmss, asspeschiofiwcnLiSnUF-liigkueresco2mBplaenxdes3waintdh to our designated tetraloop sequence and nearly ideal pril 2
GNRA tetraloop geometry as described (28). The 0
a-rPeptides, as shown in Figure 2B. Components of the tetraloop sequence from this substructure was mutated 19
a-PTC were assembled in silico, in vitro and in vivo and
from GCAA to GUAA using SYBYL-X 1.2 (Tripos
these hypotheses tested by various modeling, footprinting
International, St. Louis, MO, USA). The VMD 1.8.7
and binding assays. A longer-range goal is determining if
superposition tool (29) was used to find the best docking
a-rRNA in association with a-rPeptides can form a func-
position between each stem loop and the termini of the
tional a-PTC, as defined by catalytic activity.
helices remaining after the shaving process (see Supple-
mentary Materials for further information on the
docking process).
MATERIALS AND METHODS
Minimization
Computer modeling of a-PTC
After addition of the stem loops to the a-rRNA in silico
Model building model, the structure was manually inspected for steric
ThecrystalstructureoftheribosomefromT.thermophilus clashes and unfavorable backbone conformations. The
(PDB2J01)wasusedasthestartingstructureforbuilding solvent accessible surface areas of the stem loops were
3376 NucleicAcidsResearch,2013,Vol.41,No.5
calculatedusingaVMDtooltoensurethatallstemloops 50mM Tris–HCl pH 8.0, 100mM KCl, 33mM MgCl ,
2
were located on the surface of the a-PTC. After minor heated at 90(cid:3)C for 30s and cooled to room temperature.
manual manipulation, the a-PTC model was energy The a-rRNA solution was diluted to a total volume of
minimized in the presence of 51 Mg2+ ions observed in 240ml with water, 10(cid:4) RNase H reaction buffer and 22U
the crystal structure to be coordinated by the a-rRNA. of RNase H (New England Biolabs) to provide a master
The solvated system was neutralized with Na+ions. The mix with 0.071pmol/ml a-rRNA, 0.092U/ml RNase H in
nucleotides directly coordinating Mg2+ ions were con- 1.43(cid:4) RNase H buffer [50mM Tris–HCl (pH 8.3),
strained to fixed positions during minimization. The 75mM KCl, 3mM MgCl and 10mM dithiothreitol].
2
a-rPeptides were free from constraint during minimiza- This master mix was split into 7ml aliquots, and 3ml of a
tion. In total, 500000 steps of conjugate gradient mini- DNA probe solution was added to each tube to give 10ml
mization were performed with NAMD (29) using the reaction volumes. Each reaction contained a final 1:30
CHARMM22 force field (30). Structure validation of the RNA to DNA molar ratio. RNase H reactions were
energy minimized a-PTC model was performed using the carried out at 37(cid:3)C for 30min and quenched by quick D
o
ADIT Validation Server (http://deposit.rcsb.org/validate/ freezing. Reaction products were run on a 5% denaturing wn
), which returned no significant stereochemical issues. An polyacrylamide gel (8M urea), and imaged on a General loa
d
analysis of the structure of the a-PTC 3D model can be Electric Typhoon Trio+Imager.Tocalculatethe extentof e
d
found in the Supplementary Material. digestioninthepresenceofeachprobe,bandsrepresenting fro
the digested and undigested a-rRNA were quantified m
h
Synthesis of a-rRNA (Supplementary Figure S4). ttp
s
The DNA gene encoding a-rRNA was constructed by re- SHAPE reactions ://a
cursive PCR (31) (Supplementary Figure S2), and its ca
sequence confirmed. The rRNA fragments were linked Selective 20-hydroxyl acylation analyzed by primer exten- de
m
together at the DNA level with 11 stem loops. The sion (SHAPE) methods were adapted from published ic
RNA product of the in vitro transcription reaction runs protocols (32). In vitro-transcribed a-rRNA was .ou
p
asatightbandonadenaturinggel(SupplementaryFigure prepared in TE buffer (10mM Tris–HCl, 1mM EDTA, .c
o
S3). Gene synthesis and characterization, and RNA syn- pH 8.0) at 100ng/ml a-rRNA. Thirty-two microliter m
thesisbyinvitrotranscriptionaredescribedindetailinthe aliquots of the RNA solution were added to 4ml of 10(cid:4) /na
Supplementary Materials. folding buffer (500mM NaHEPES pH 8.0, 2M NaOAc, r/a
varying MgCl2) and incubated at 37(cid:3)C for 20min. rticle
RNase H cleavage reactions -a
NMIA modification of a-rRNA b
s
Oligodeoxynucleotide probes A 130-mM N-methylisatoic anhydride (NMIA; Tokyo tra
c
DNA probes for RNase H cleavage reactions were Chemical Industry Co., Ltd.) solution in 2ml dimethyl t/4
obtained from Integrated DNA Technologies, Inc. or sulfoxide (DMSO) was added to solutions of 18ml 1/5
EurofinsMWGOperon.Thenomenclaturefordescribing annealed a-rRNA. Control reactions contained DMSO /3
3
the DNA probes indicates if the RNA target is predicted only. Reactions were carried out for 1h at 37(cid:3)C. For 73
to be double-stranded (D), single-stranded (S) or a stem- NMIAmodificationunderdenaturingconditions,thereac- /24
1
loop (T), and gives the helix number that either contains tions were run at 90(cid:3)C for 4min. NMIA-modified RNA 4
9
orsucceedsthetargetsite.TheDNAprobesare:50-TTTC was purified from reaction mixtures with the Zymo RNA 07
GGGTCC-30(S32),ACCAGCTATC(S36),CATTCGCA Clean+Concentrator-25 kit (Zymo Research). a-rRNA by
CT(S26),CGTTACTCAT(S61),TGCAGAGTTC(S62), waselutedwith25mlTEbuffer.Recoveryafterpurification gu
e
GTTCAATTTC (S72),GGTCTTTTCG (S74),CCTGTT was >75%. s
ATCC (S89), ACATCGAGGT (S90), TCTGAACCCA t on
(S93), GATAGAGACC (S73), GCCTTACGGC (T), G Reverse transcription of NMIA-modified a-rRNA 05
CCAGGGCTA (D33), CCCTCGCCGA (D61) and AG Four different 50-[6-FAM]-labeled DNA oligonucleotides A
p
CTCCACGG (D74). Nucleotide numbers targeted by (EurofinsMWGOperon)wereusedasprimers(eachhelix ril 2
each probe are provided in Supplementary Table S1. in parenthesis either contains or succeeds the primer 0
1
binding site): 50-TGCCCGTGGCGGATAGAGAC-30 9
RNase H reaction (helix 73), 50-ACATCGAGGTGCCAAACCGCC-30
In vitro-transcribed a-rRNA was dephosphorylated with (helix 89), 50-GTTCAATTTCACCGGGTCCCTCG-30
Antarctic Phosphatase (New England Biolabs). Ten (helix 61) and 50-CGTTACTCATGCCGGCATTCGC-30
picomoles of the a-rRNA was 50-end labeled with (helix 26). Modified RNA (20ml) was added to 8pmol of
0.05mCi of [g-32P]ATP using T4 polynucleotide kinase each primer in 10ml of TE buffer. To anneal primers,
for 30min at 37(cid:3)C. The end-labeled RNA was separated samples were heated at 95(cid:3)C for 1min, held at 65(cid:3)C for
from unreacted [g-32P]ATP with Ambion NucAway spin 3min and then placed on ice. SuperScript III Reverse
columns. Divalent cations were removed from the labeled Transcriptase (Invitrogen) was used in reverse transcrip-
a-rRNA by heating at 90(cid:3)C for 2min in the presence of tion (RT) reactions. RT buffer (19ml) was added at 30(cid:3)C
Divalent Cation Chelating Resin (100–200 Mesh sodium to yield 50mM Tris–HCl pH 8.3, 75mM KCl, 3mM
form; Hampton Research). To anneal the a-rRNA, MgCl ,2mMDTTand 250mM ofeachdNTP(final con-
2
17pmol of unlabeled a-rRNA was mixed with trace centrationsin50ml).RTmixtureswereheatedat55(cid:3)Cfor
amounts of end-labeled a-rRNA, dissolved in 24ml of 1min before addition of 1ml Superscript III Reverse
NucleicAcidsResearch,2013,Vol.41,No.5 3377
Transcriptase enzyme mix (200U). Reactions were Gel shift assays with a-rRNA and a-rPeptides
incubated at 55(cid:3)C for 2h and terminated by heating at
Cloning of MBP-a-rPeptide fusion proteins
70(cid:3)C for 15min. A sequencing control reaction of un-
DNA genes encoding a-rPeptides L2, L3, L15 and L22
modified in vitro-transcribed RNA was dissolved in TE
(Table 1) were synthesized by recursive PCR as described
buffer at 31ng/ml. Aliquots of RNA solution (20ml) were
earlier (31). a-rPeptide genes were ligated into the
annealed to the DNA primers as described earlier. RNA
pMAL-c5x vector (New England Biolabs) downstream
was sequenced by RT/chain termination using all four
of the malE gene, which encodes for maltose-binding
dideoxynucleotidetriphosphates (ddNTPs), at a ratio of
protein (MBP). Each resultant MBP fusion protein bears
8:1 ddNTP to dNTP, and a control reaction without
an a-rPeptide (Table 1) on the C-terminus and is referred
ddNTPs was also prepared.
to here as an MBP-a-rPeptide. Information on gene se-
quences, cloning, expression and purification of MBP-a-
Capillary electrophoresis of RT reaction products
rPeptide fusions is provided in the Supplementary
One microliter of RT reaction mixture was mixed with D
Materials. ow
0.3ml of ROX-labeled DNA sizing ladder (for align- n
ment of disparate traces) and 8.7ml Hi-Di Formamide a-rRNA/MBP-a-rPeptide interactions loa
d
(Applied Biosystems) in a 96-well plate. Plates were e
A solution of 1mM a-rRNA was prepared in 20mM d
heated at 95(cid:3)C for 5min and the products were resolved Tris-Cl, pH 8. The a-rRNA solutions were heated at fro
b(AypcpalpieildlarByieolseycsttreomphs)oraetsis6u5s(cid:3)iCngwai3th130aGceunsetotimcAflnuaolyrzeesr- t8o5(cid:3)C30(cid:3)fCor. 3A0ts caonndcecnotorlaetdiolninseaorfly1–a1t0amMrat,eeoafch1.5M(cid:3)CB/Pm-ain- m http
cloeandceedswpietchtrPaelrfcoarlmibarnactieonO.ptTimhiezedcaPpoilllyamryer-a4rr(aAyppwlieads rfoPreptviadreiafbulseionduwraastiionncsub(asteevderwailthh1omuMrs at-orRNovAernatig4h(cid:3)tC). s://ac
a
Biosystems). Interactions between the MBP-a-rPeptides and a-rRNA de
m
were analyzed on a 5% native PAGE gel with 3% ic
SHAPE data processing glycerol. Gels were visualized using a two-color fluores- .ou
SHAPE data were processed as described earlier (33). p
cence dye protocol (36). .c
Several nucleotides were excluded from SHAPE analysis o
m
because the reverse transcriptase gave high background /n
Yeast three-hybrid assays a
terminationinthe((cid:5))NMIAreactions.Thesenucleotides r/a
were determined statistically [termination value (cid:6)90% of Hybrid cloning rtic
the (+) NMIA value, where both values exhibit (cid:6)0.25 The genes of T. thermophilus ribosomal proteins L2 le
-a
normalized intensity]. (NCBI accession number NC_005835: c1265055-1264225), b
s
L3 (NC_005835:c1266643-1266023), L4 (U36480), L15 tra
c
(NC_005835:c1258456-1258004) and L22 (NC_005835: t/4
Gel mobility of a-rRNA c1263932-1263591) were amplified from genomic DNA 1/5
The a-rRNA was first treated with divalent chelating and individually cloned into the pACTII vector (37). /33
AmplificationprimersequencesaregiveninSupplementary 7
beads (Divalent Cation Chelating Resin, 100–200 Mesh 3
sodium form; Hampton Research) to remove Mg2+ions. Table S3. In the pACTII vector, ribosomal protein genes /24
1
a-rRNA in 10mM Tris-Cl, pH 8, was mixed with the were fused to the C-terminal end of the GAL4 transcrip- 49
chelating beads and heated at 90(cid:3)C for 2min. The tional activation domain (GAD). The a-rRNA gene was 07
amplified from the transcription vector described in the b
mixture was then chilled on ice for 20min. The a-rRNA y
Supplementary Materials and cloned into the T-cassette g
was separated from the beads and buffer with a 0.2-mm u
vector (38). The a-rRNA gene was ligated to the 50-end of es
cw8e5en(cid:3)rCteriaffoudrgda3eld0fistolatenTrdE(aAlsloomlwuicetoidonnt)o.scAoofloivqlauarotytrisnogoomf[M1te8gm02+np],gerhaaet-aurtRreedNfoaArt thphyreobtrgeiidenneph5ey0nb-cMroiddSi2nGgcAflooDnr-epMd53Sin2tcoRloNntheAde. TPino-tcsoaitsisvpeetAtecCoTnvIterIcotol(r3R8Na,3n9Ad) t on 05 A
25min before loading on a 5% native-PAGE gel. p
were a generous gift from Dr James Maher. ril 2
0
1
In vivo binding assays 9
a-rRNA/a-rPeptide L4 interaction by continuous variation
Yeast three-hybrid assays were performed in the YBZ-1
analysis
yeast strain as described earlier (37,40,41). Double
a-rPeptide L4 (Table 1) was purchased from Peptides transformants with the protein and RNA-hybrid con-
International (Louisville). In a series of solutions, the total structs were selected in medium lacking adenine and
concentration ([a-rRNA]+[a-rPeptide L4]) was held leucine (CM-AL). Interaction of RNA with protein
constantat60mM,whilethemolefractionsofthetwocom- resultsinactivationoftheGAL4promoterandexpression
ponents were varied from 0.0 to 1.0 (34,35). The mixtures of LacZ. The strength of an RNA-protein interaction is
were prepared in 10mM Tris-Cl buffer, pH 8, heated at indicatedquantitativelybyb-galactosidase(b-gal)activity
85(cid:3)C for 30s and annealed by cooling to 30(cid:3)C at a linear (42). The positive control used hybrids p50-MS2 and
rateof1.5(cid:3)C/min.Theannealedsamplesweretransferredto GAD-p53, which are known to interact strongly in the
amicroplate.Thefluorescencesignalofthetryptophancon- yeast three-hybrid system (39). Negative controls include
tained in a-rPeptide L4 was measured at 334nm at 25(cid:3)C each protein hybrid with the MS2 RNA only (T-cassette
using a Biotek Synergy H4 Multi-Mode Plate Reader. vector without insert). Growth observed in negative
3378 NucleicAcidsResearch,2013,Vol.41,No.5
controls was considered to represent background signal.
All experiments were performed in a minimum of six
replicates.
RESULTS
a-PTC in silico
The a-PTC was modeled in three dimensions (Figure 2B).
To form the in silico a-PTC model, the X-ray structure of
the LSU of T. thermophilus (PDB 2J01) was shaved to a
sphere of around 30A˚ , and 12 of the 13 resulting rRNA
fragments were stitched together with stem loops, each of D
o
sequence50-gccGUAAggc-30.EachpairofadjacentrRNA wn
fragmentswasconnectedwithastemloopasdescribedin loa
d
the Supplementary Materials. An exception is the rRNA e
d
fragments adjacent to helices 47 and 61, where the rRNA fro
fragments were joined directly, without a stem loop m
h
(Figure 2A). The completed a-rRNA is composed of ttp
5cl0e5otniduecslefortoimdes11desrtievmedloforopms.Tthhee2a3-SPTrRCNinAsiplilcuosc1o1n0taninus- s://ac
a-rRNA (615 nucleotides), 51 Mg2+ ions and five ad
e
a-rPeptides (Table 1). m
ic
In the 3D model, the components that are common .o
u
between the a-PTC and the T. thermophilus LSU crystal p
.c
structure have conserved conformation and interactions. o
m
The stem loops are constrained to canonical GNRA /n
a
tetraloop conformation (28) and are located on the r/a
surface of the a-PTC. The stem loops do not engage in rtic
unfavorablestericcontactswithotherpartsofthea-PTC. le
-a
The model is stereochemically reasonable. b
s
The predicted secondary structure of the a-PTC shown tra
c
inFigure2Aisbasedonthecanonicalsecondarystructure t/4
of the 23S rRNA. There are some small differences 1/5
between the canonical secondary structure and the /3
3
actual secondary structure found in the crystal structure. 73
These difference are responsible for some otherwise unex- /24
1
pected experimental observations, as discussed below. 4
9
0
7
b
RNase H characterization of a-rRNA secondary structure y
g
u
RNase H and 15 different DNA oligonucleotide probes Figure 3. Probing the secondary and tertiary structure of a-rRNA. es
were employed to characterize the secondary structure of (A) SHAPE and RNase H mapping. Red triangles mark SHAPE t o
a-rRNAinvitro(Figure3A).RNaseHcleavageisbroadly reactivities in 250mM Na+, mapped onto the predicted secondary n 0
structureof a-rRNA.Larger triangles indicategreater SHAPE reactiv- 5
used to characterize RNA secondary structure, allowing ity.RNaseHDNAprobesareindicatedbygreenlines.Circlesindicate A
p
dstirsacnridmeidnaRtiNonA (b4e3t,w44e)e.nRNsiansgeleH-strreacnodgendizeasnadnddcoleuabvlees- ehxatlef-nfitlloedf RcNircAlesdi(gbeesttwioenenby25RNanadse7H5%: fi)lleadndcirecmlepsty(mcoirrceletsha(n<2755%%))., ril 20
RNA/DNA heteroduplexes; double-stranded RNA (B) Effects of 10mM Mg2+ on SHAPE reactivity suggest formation 19
of tertiary structure. Green triangles show the greatest increases in
inhibits cleavage by blocking heteroduplex formation. SHAPE reactivity upon addition of Mg2+. Blue triangles show the
The RNase H cleavage pattern of the a-rRNA shows greatestdecreasesinreactivity.(C)MultidentateMg2+-phosphateinter-
good overall agreement with predictions of the in silico actions observed in the T. thermophilus LSU (PDB 2J01) are mapped
model, suggesting formation of the secondary structure onto the predicted secondary structure of a-rRNA. Magenta circles
indicate first-shell Mg2+-OP (magnesium-phosphate oxygen) inter-
shown in Figures 2A and 3. Four DNA probes were
actions. Magenta lines indicate PO-Mg2+-OP linkages. Gray shading
designed to target regions of a-rRNA that are double- in panels A and B indicates rRNA where SHAPE data were not ac-
stranded in the predicted secondary structure, and so cessible. SHAPE reactions were performed in 50mM NaHEPES, pH
should not yield RNase H cleavage. Eleven DNA probes 8.0, 200mM NaOAc, 0 or 10mM MgCl2.
were designed to target regions of a-rRNA that are
single-stranded in the predicted secondary structure, double-stranded target regions are protected from
whichshouldyieldRNaseHcleavage.Tenof11predicted cleavage, or showed only partial cleavage.
single-stranded RNA target regions are cleaved partially RNase H cleavage in the presence of two of the DNA
or fully by RNase H. Three of the four predicted probes initially appeared to suggest inconsistencies
NucleicAcidsResearch,2013,Vol.41,No.5 3379
between the predicted and observed a-rRNA secondary from 25 to 37%. The smallest triangles indicate the 109
structure, but further analysis indicated otherwise. nucleotides with reactivities from 8 to 24%. Nucleotides
Specifically,probeS36targetsRNAthatissingle-stranded with reactivities of (cid:7)7% are considered to be unreactive.
in the predicted secondary structure, and therefore was Full numerical SHAPE values are available in the
anticipated to yield cleavage by RNase H. However, in- Supplementary Material.
spection of the T. thermophilus 50S crystal structure (21) Of the 146 nucleotides that are reactive to the SHAPE
reveals that the target region of probe S36 is engaged in reagent, 133 (91%) fall in regions expected to be
secondary interactions not reflected in the canonical 23S single-stranded, in that they do not form base pairs in
secondary structure on which the predicted a-rRNA sec- the predicted secondary structure. These nucleotides are
ondary structure is based. S36 does not yield cleavage of inloops,bulgesorothersingle-strandedregions.Thereso-
a-rRNA consistent with the secondary interactions lutionofSHAPEissufficientlyhightoallowidentification
observed in the LSU crystal structure. of small bulges such as those in helices 35, 61, 64, 73, 74
Oligonucleotide D74 targets Helix 74, which is double- and89(Figure3A).Thirteenreactivenucleotidesarebase Do
w
stranded in the predicted secondary structure, and thus pairedinthepredictedsecondarystructure.Thesenucleo- n
D74 was not expected to yield cleavage. Contrary to this tides represent <6% of the 232 nucleotides that are loa
d
prediction, D74 yields significant cleavage ((cid:2)88%) expected to be double-stranded, the remaining 94% of e
d
compared with the other double-stranded target regions. which exhibit low SHAPE reactivity as predicted. The fro
Comparing RNA/RNA versus DNA/RNA duplex free 133 nucleotides that SHAPE identifies as flexible m
h
energies (45,46), we find that the stability of Helix 74 compose 59% of the 227 single-stranded nucleotides in ttp
h((cid:5)et5e.r8o0dkucpalle/xmoflo)rmisedlesbsythDan74thwatithofthtehetaDrgNeAt /RRNNAA the predicted secondary structure. s://ac
a
((cid:5)12.5kcal/mol). This differential stability may allow gccGUAAggc stem-loops fold in all contexts d
e
the probe to invade the RNA/RNA duplex, accounting SHAPE data were obtained for 106 (96%) of the 110 nu- m
ic
for the cleavage. cleotides within the 11 stem loops. The stem regions are .o
u
unreactive. Only 5 of the observable 62 nucleotides in the p
.c
stemregions(8%)exhibitreactivity,whichislowforeach. o
SHAPE characterization of a-rRNA secondary structure m
The loop regions yield a consistent pattern of reactivity /n
SreHacAtsPEwiuthtil2iz0-ehsyadnroexlyelctgrroopuhpilse,ofinRtNhiAs c(a4s7e).NSMHAIAP,Ethraet- tthhaet1i1ssintedmepleonodpesn,tthoefUthAeAsunrruoculenodtiindgesseaqreuemnocer.eIrnea1c0tivoef ar/artic
activity is modulated by RNA flexibility. Base-paired nu- thantheprecedingG.Thispatternofreactivityisconsist- le
cleotides exhibit low reactivity as their flexibility is entwithknownpatternsofflexibilityofGNRAtetraloops -ab
s
constrained. Single-stranded nucleotides are flexible and (28,52).NucleotidesNRAofaGNRAtetralooparemore tra
reactive to the electrophile. SHAPE has been shown to polymorphic and flexible than the G. The N nucleotide is ct/4
be accurate for mapping secondary structure of many the most polymorphic of all. 1/5
RNAs, including Escherichia coli rRNA (48). TheSHAPEresultssuggestthatthestem-loopreplacing /3
3
The SHAPE data show excellent correspondence with helix91(Figure3A)mightnotformthepredictedsecond- 73
thepredictedsecondarystructureofa-rRNA(Figure3A). ary structure. The stem loop at this position exhibits a /24
These data were obtained by probing the a-rRNA in the distinctive pattern of reactivity. Two nucleotides in the 14
presence of monovalent cations (250mM Na+) and the stem appear to be anomalously flexible. This SHAPE 907
absence of Mg2+. These conditions are known to data will inform design changes in future iterations of by
support the formation of RNA secondary structure, but a-rRNA. gu
nottertiarystructure(49–51).SHAPEdatawereobtained e
s
for 459 of the 505 nucleotides (91%) of the a-rRNA t o
SHAPE characterization of a-rRNA tertiary interactions n
derived from the 23S (stem-loop nucleotides are 0
5
analyzed separately below). The values and statistics RNAs require Mg2+ ions to form compact structures A
p
pthreosveidneducilneotthideesremdeariinvdederforofmthisthseec2ti3oSn rrRefNerAo.nDlyattoa (r4ic9h–5i1n).MThg2e+coiornesof(9t,2h2e,5a3s)s.emInbleodurLS3UD imsopdaerlticouflatrhlye ril 20
1
were not obtainable for RNA near the termini, or for a a-PTC, the conformation of the a-rRNA is stabilized in 9
few nucleotides with high background (i.e. high reverse part by Mg2+ ions (Figures 2B and 3C) and also by
transcriptase termination in samples not treated with tertiary interactions between RNA elements that are
NMIA). These regions are shown in gray on the remote in the primary sequence. Here, we ask if a-rRNA
a-rRNA secondary structure in Figure 3A. in vitro, upon the addition of Mg2+, forms a compact
Nucleotides were binned into four groups according to structure consistent with the in silico a-PTC model
their normalized SHAPE reactivity. U2574 (NCBI E. coli (Figure 2B).
numbering) is the most reactive nucleotide, with an A network of phosphate-Mg2+-phosphate interactions
absolute reactivity of 3.24 (arbitrary units), and is con- is anticipated in the a-PTC, based on the network in the
sidered 100% reactive. Bin assignments are indicated by LSU crystal structure (PDB 2J01) (22). This phosphate-
thesizeofredtrianglesinFigure3A.Thelargesttriangles Mg2+-phosphatenetworkisindicatedinFigure3C,where
denote the 13 nucleotides with normalized SHAPE magenta circles represent nucleotides with phosphate
reactivities ranging from 38 to 100%. Triangles of inter- oxygens that make direct contacts with Mg2+ ions
mediate size indicate the 24 nucleotides with reactivities (<2.4A˚ cutoff distance). Long-range phosphate-Mg2+-
3380 NucleicAcidsResearch,2013,Vol.41,No.5
phosphate linkages are indicated by magenta lines. The a-rRNA appears to have one predominant conformation
proposed network includes all Mg2+ ions that interact and an ensemble of minor conformations (Figure 4),
with phosphates of two or more of the nucleotides. inferred from the smeared intensity between the lower
Formation of the phosphate-Mg2+-phosphate network is band and fainter upper band on the native gel. The
coupled with formation of base-base tertiary interactions. smear does not arise from heterogeneity in length, as
Therefore, Mg2+is expected to influence the rRNA flexi- a-rRNA runs as a tight single band on denaturing gels
bility and SHAPE reactivity of nucleotides that contact (Supplementary Figure S3). During the course of Mg2+
Mg2+or are involved in long-range tertiary interactions. titration, the a-rRNA is seen in a native gel to transition
This pattern of Mg2+-dependent SHAPE reactivity has toasingle,slower-runningstate(Figure4).Thistransition
previously been observed for tRNA, RNase P, the P4-P6 appears complete at 250mM Mg2+.
domain of the Tetrahymena Group I intron and Domain
III of the 23S rRNA (33,47,54,55). a-rRNA forms a specific 1:1 complex with a-rPeptide L4
The influence of Mg2+ on a-rRNA SHAPE reactivity The fluorescence of the tryptophan within a-rPeptide L4 Do
(fFolidgiunrge.3SBo)mseuMggges2+ts-itnhdautcMedgc2+hadnogeessiinndSeHedAiPndEurceeacgtliovbitayl (aTnadbale-r1P)epwtaidseuLse4ditnoamCoonnittoinruthoeusinVtearraiacttiioonnoexfpae-rriRmNenAt wnload
of a-rRNA are clustered around hypothesized regions of e
(34,35).Thetwospeciesweremixedinaseriesofsolutions d
direct RNA-Mg2+contact similar to those in the folded of varying mole fractions. The total concentration of the fro
LSU. Others are observed in regions of hypothesized m
two species was held fixed throughout. Continuous vari- h
long-range tertiary interactions. Absolute SHAPE data ation of a-rRNA and a-rPeptide L4 shows a distinct in- ttp
aicraelgdraaptaheadreinctohnetSauinpepdleminenataSryupFpilgeumreenSt5a.ryThEexnceulmfierle- flection at 1:1 stoichiometry (Figure 5), where the molar s://ac
a
(Supplementary Dataset 1). d
e
Changes in SHAPE reactivity upon addition of Mg2+ m
ic
(10mM) were determined. The greatest changes in .o
u
SHAPE reactivity (>40% change) are mapped onto the p
.c
predicted secondary structure of a-rRNA (increases: om
green; decreases: blue, Figure 3B). Nucleotides with /n
a
large changes in reactivity are dispersed throughout the r/a
sequence (Supplementary Figure S5) and the secondary rtic
structure. Nucleotides for which the absolute SHAPE re- le-a
activity is small ((cid:7)0.3) were omitted from the analysis to b
avoidattributing artificial significance tosmall changes in MFiggu2+rein4d.uTcthieonefofefcftooldfinMg.g2S+hoownngehlermeoibsiali-tryRoNfAthaenan-eraRleNdAinsu1g0gmesMts strac
absolute value. Tris, pH 8.0, and varying [Mg2+], resolved on a 5% native acrylamide t/4
1
Of the 65 rRNA nucleotides (excluding stem-loops) gel.Lane1,[Mg2+]=0mM;Lane2,12.5;Lane3,25;Lane4,50;Lane /5
with altered SHAPE reactivities upon addition of Mg2+ 5, 100; Lane 6, 250; Lane 7, 500. /3
3
7
(Figure 3B), 25 (38%) are within three residues of a nu- 3
cleotide that contacts a Mg2+ion in the predicted inter- /24
1
action network in Figure 3C. Most of the other Mg2+ 4
9
0
effects are at or near bulges and loops, assumed to be 7
b
involved in tertiary interactions. The data are consistent y
with induction by Mg2+of local and long distance inter- gu
e
actionsthatalternucleotideflexibility.Theresultssuggest st o
a transition of the a-rRNA from an extended secondary n
0
structure to a collapsed structure with tertiary 5
A
interactions. p
The SHAPE reactivities argue against significant alter- ril 2
ation of the secondary structure upon addition of Mg2+. 01
9
Only a smallnumberofhelicalnucleotidesshowMg2+-de-
pendent changes. Of the 65 rRNA nucleotides that show
significant Mg2+ effects, only a few (9 nucleotides) are
foundinregionsthatarepairedinthepredictedsecondary
structure, and several of these are adjacent to predicted
bulges or other single-stranded regions. Changes in
Figure 5. a-rRNAanda-rPeptideL4areassemblycompetentandform
chemical reactivity upon folding in the presence of acomplexwith1:1stoichiometry.Fluorescencesignalat334nmisfrom
rProteinsora-rPeptideswillbeinvestigatedinfuturework. the tryptophan residue of the a-rPeptide L4. In this plot of a continu-
ous variation experiment of a-rPeptide L4 with a-rRNA, X and
L4
X values on horizontal axis denote mole fraction of the peptide
Magnesium alters the gel mobility of a-rRNA a-rRNA
and RNA in each sample, where the total concentration
([a-rRNA]+[a-rPeptide L4]) was held constant at 60mM. The discon-
The SHAPE data support the hypothesis that the con-
tinuity at equivalent mole fractions of peptide and RNA indicates a
formationofthea-rRNAisalteredbyMg2+.Thishypoth-
complex with 1:1 stoichiometry. These binding assays were performed
esisisalso testedbygelmobility. In theabsenceof Mg2+, in 10mM Tris, pH 8.0, at 25(cid:3)C.
NucleicAcidsResearch,2013,Vol.41,No.5 3381
fractionsofa-rPeptideL4anda-rRNAareboth0.5.Thus, form a complex, they co-localize on the gel, producing a
continuous variation of a-rRNA and a-rPeptide L4 yellow band (Figure 6).
suggests a 1:1 stoichiometry of binding. In a control ex- The results indicate that MBP fusions with a-rPeptides
periment, an inflection was not observed for the continu- L3,L15orL22formcomplexeswitha-rRNAintherange
ous variation of the P4-P6 domain of the Tetrahymena of1–10mMoftheMBP-a-rPeptidefusion(Figure6).Each
Group I intron with a-rPeptide L4 (Supplementary of these three MBP-a-rPeptide fusions shifts the a-rRNA
Figure S6). band on the gel. MBP-a-rPeptide L3 required overnight
incubation to form a complex with a-rRNA, but MBP-a-
a-rRNA forms complexes with MBP-a-rPeptides rPeptides L15 and L22 required only a few hours incuba-
L3, L15 and L22 tion. MBP-a-rPeptide L2 did not form a complex with
Theinteractionsbetweena-rRNAanda-rPeptidesL2,L3, a-rRNA under any of the conditions tested, even at con-
L15 or L22 are not detectable by fluorescence due to the centrations an order of magnitude greater than those suf-
D
absence of fluorescent amino acids in these peptides. ficient for binding of MBP-a-rPeptide L3, L15 or L22. o
w
Instead, the interactions of a-rPeptides L2, L3, L15 or Here, we assayed a-rRNA interactions with a-rPeptides nlo
L22 with a-rRNA were evaluated by electrophoretic in the absence of Mg2+. The a-rRNA exhibits two ad
mobility shift assay (EMSA). The peptides alone are too dominant conformations on EMSA gels (Figure 6) ed
small to cause a visible change in a-rRNA gel mobility. indicated by two bands. The binding of any a-rPeptide, fro
m
Therefore,thea-rPeptideswerefusedtotheC-terminusof like binding of Mg2+, induces formation of a single state, h
MBP using molecular cloning techniques. In a two-color indicated by a single band on the gel. A control ttp
s
EMSA(36),uncomplexedRNAisgreen,andfreeprotein experiment shows MBP alone does not bind to a-rRNA ://a
(MBP-a-rPeptidefusions)isred.IfanRNAandaprotein (Figure 6A).At highprotein concentration(>50mM),the ca
d
e
m
ic
.o
u
p
.c
o
m
/n
a
r/a
rtic
le
-a
b
s
tra
c
t/4
1
/5
/3
3
7
3
/2
4
1
4
9
0
7
b
y
g
u
e
s
t o
n
0
5
A
p
ril 2
0
1
9
Figure 6. Gel shift analyses of interactions between a-rRNA and MBP-a-rPeptide fusions. RNA, protein and RNA–protein complexes were
visualized on 5% native-PAGE gels by two-color EMSA. All binding reactions were performed with 1mM a-rRNA, in 20mM Tris buffer, pH
8.0.Ineachgel,thefarleftlanescontaineda-rRNAonly(noprotein).(A)Inacontrolassay,a-rRNAdoesnotinteractwithMBPalone:a-rRNA
incubatedwithMBP(lefttoright:0,1,2,4,8,10,50and100mM).(B)a-rRNAincubatedwithMBP-a-rPeptideL3(lefttoright:0,1,2,4,8and
10mM). (C) a-rRNA incubated with MBP-a-rPeptide L15 (left to right: 0, 1,2, 4, 8and 10mM). (D) a-rRNA incubated with MBP-a-rPeptide L22
(left to right: 0, 1, 2, 4, 8 and 10mM). (E) a-rRNA incubated with MBP-a-rPeptide L2 (left to right: 0, 1, 10, 50 and 100mM).
3382 NucleicAcidsResearch,2013,Vol.41,No.5
a-rRNA is seen to degrade, as indicated by faint low-mo-
lecular weight RNA bands on the gels.
Using the method of Williamson (56), we have
estimatedthedissociationconstants(K )ofthecomplexes
d
formedbyMBP-a-rPeptidesL3,L15orL22witha-rRNA.
Data were obtained by integrating the band intensities
from the EMSA gels (Figure 6). MBP-a-rPeptide L3
binds weakly, and the K is estimated to be >10mM.
d
The K is 4.6mM for MBP-a-rPeptide L15 and 7.4mM
d
for MBP-a-rPeptide L22. Kimura previously measured a
K of 1mM fortheinteraction of intact ribosomal protein
d
L2 with appropriate fragments of the 23S rRNA (57).
Hachimori measured a K of 10nM for the interaction D
d o
w
of intact ribosomal protein L3 with the Sarcin/Ricin n
Domain of 23S rRNA (58). Nierhaus and others used loa
d
filter binding and sedimentation to determine e
d
stoichiometries of binding of ribosomal proteins to fro
rRNA (59). Ribosomal proteins L2, L3 and L4 are m
h
classified as normal binders to the 23S rRNA in the ttp
pwreeaskenbciendoefrs4.mM Mg2+. L15 and L22 are classified as s://ac
a
d
e
a-rRNA interacts with intact rProteins L3, L4, L15 and m
ic
L22 in vivo .o
u
p
To determine if our in vitro results could be replicated .c
o
invivoweusedtheyeastthree-hybridsystemtoinvestigate m
/n
interactions of a-rRNA with a-rPeptides and intact a
rProteins. The interactions of full length rProteins L2, r/a
L3,L4,L15orL22witha-rRNAwerecharacterized(sche- rtic
le
matic provided in Figure 7A). Each rProtein was fused to -a
b
theCterminusoftheGAD,whichinteractswiththeLacZ s
tra
reporter gene. These hybrids are referred to as GAD-L2, c
GAD-L3, GAD-L4, GAD-L15 and GAD-L22. The t/4
1
RNA–rProteininteractionswereassayedbyquantification /5
/3
of increased b-gal activity due to enhanced expression of 3
7
3
theLacZreportergene(Figure7B).Bythisassay,a-rRNA Figure 7. (A) Schematic of the yeast three-hybrid assay used to char- /2
exhibited the strongest in vivo interaction with L4, with a acterize interactionsbetweena-rRNAandribosomalproteins.Inyeast 41
strain YBZ-1, the LacZ reporter gene is controlled by the LexA 4
b-gal average activity of 1150Miller Units (MU). The 9
operator. Hybrid 1, a LexA/MS2 binding protein fusion, binds to the 0
second strongest in vivo interaction with a-rRNA was DNA binding site. The MS2 coat protein domain binds tightly to the 7 b
observedwithL22at750MU,andmoderateinvivointer- MS2 RNA, which is fused to the RNA sequence of interest (e.g. y g
actions were observed with L15 (420MU) and L3 a-rRNA).TherProteinofinterestisfusedtotheyeastGAL4transcrip- ue
(400MU). Consistent with the in vitro assays, a-rRNA tinionexaplraecstsiivoantioonf tdhoemLaaincZ(GreApDor)t.eIrngveinveo,RwNhiAch-pirsotqeuinanbtiifineddingbyrebs-uglatsl st on
did not exhibit any measurable in vivo interaction with activity. (B) In vivo interactions between a-rRNA and individual 0
5
L2. With a similar experimental approach, interactions rProteins. Interaction is quantified by b-gal activity, reported in MU. A
wOeurre cnuortreonbtsehrvyepdotbheetswiseeins tthheata-trhPeepltinidkeesrabnedtwa-ereRnNtAhe. IGnAteDra-cLti3o,nGs AwDe-rLe4,asGsaAyDed-L1b5etwanedenGAa-DrR-LN2A2-M(bSla2ckabnadrs)G. APoDsi-tLiv2e, pril 2
a-rPeptide and GAD may need to be lengthened to allow cNoengtarotilvewcaosntrRoNlsAconaspitsatemderofpM50S-M2 SR2NAandandGtAhDe-ipn5d3ica(tgerdayprobtaeri)n. 019
and observe interactions. Further work in this area is in hybrid (white bars).
progress.
DISCUSSION
an effort to exploit the ribosome as a tool for learning
Translation and the ribosome are some of the most about ancient biology. Ancient components of the LSU,
illuminating biochemical links between current and asinferredfromconsensusamongmodelsofearlyriboso-
ancient biological systems (60). Translation provides a mal evolution, were constructed and characterized. The
powerful experimental and theoretical system for conclusion here is that the core of the LSU has retained
exploring life’s oldest biological processes and molecules an ancient ability to fold and assemble over vast evolu-
(2–7). Some parts of the ribosome are thought to predate tionary time frames of billions of years. Relatively small
our current biology of DNA, RNA and coded protein. ancestral components of the LSU are folding competent
Following previous work (10,61–64), we have undertaken and assembly competent.