DNA-binding proteins and evolution of transcription regulation in the archaea

13 Pages·1999·1.08 MB·English

4658–4670 NucleicAcidsResearch,1999,Vol.27,No.23 ©1999OxfordUniversityPress
DNA-binding proteins and evolution of transcription
regulation in the archaea
L. Aravind1,2 and Eugene V. Koonin1,*
1NationalCenterforBiotechnologyInformation,NationalLibraryofMedicine,NationalInstitutesofHealth,Bethesda,
MD20894,USAand2DepartmentofBiology,TexasA&MUniversity,CollegeStation,TX77843,USA
ReceivedMay3,1999;RevisedandAcceptedSeptember20,1999
ABSTRACT and archaeal/eukaryotic lineages. During subsequent
evolution,thesimilaritybetweenarchaealandbacterial
Likely DNA-binding domains in archaeal proteins
gene/operon transcriptional regulators might have
were analyzed using sequence profile methods and
been established and maintained through multiple
available structural information. It is shown that all
horizontalgenetransferevents.
archaea encode a large number of proteins
containing the helix–turn–helix (HTH) DNA-binding
domainswhosesequencesaremuchmoresimilarto INTRODUCTION
bacterialHTHdomainsthantoeukaryoticones,such
Thestudyofbacterialandphageoperonsgaverisetosomeof
as the PAIRED, POU and homeodomains. The
thebasictenetsofourunderstandingofgeneticcontrolwhich
predominantclassofHTHdomainsinarchaeaisthe includetheuseofDNA-bindingproteinstopositivelyornegatively
winged-HTH domain. The number and diversity of regulatetranscriptioninitiationbytheRNApolymerase.Since
HTH domains in archaea is comparable to that seen the determination of the crystal st(cid:1)ructures of the c1 and Cro
in bacteria. The HTH domain in archaea combines repressor proteinsofbacteriophage (1,2),the helix–turn–helix
withavarietyofotherdomainsthatincludereplication (HTH)domainhasbecomeoneoftheparadigmsofDNA–protein
interactions.AnalysisofthestructuresofseveralHTHproteins
systemcomponents,suchasMCMproteins,translation
systemcomponents,suchastheaaaa -subunitofphenyl- showedthattheycontactDNAbymeansofinsertionofathird,
distalhelixofaright-handedthree-helixbundleintothemajor
alanyl-tRNA synthetase, and several metabolic
groove of double-stranded DNA (3,4). Subsequently, a
enzymes.ThemajorityofthearchaealHTH-containing
combination of X-ray crystallography and protein sequence
proteins are predicted to be gene/operon-specific analysis of bacterial DNA-binding regulatory proteins has
transcriptional regulators. This apparent bacterial- revealed thatthe HTH domain is a common theme present in
type mode of transcription regulation is in sharp mostofthem(5–8).Studiesondevelopmentalanddifferentiation
contrasttotheeukaryote-likelayoutofthecoretran- regulators in eukaryotes have led to the identification of a
scriptionmachineryinthearchaea.Inadditiontothe number of transcription factors that possess conserved
predicted bacterial-type transcriptional regulators, the domains, such as the homeodomain, the Paired domain, the
POU domain and the Myb/SANT domains. Sequence–structure
HTH domain is conserved in archaeal and eukaryotic
studiesindicatethatalthoughtheseproteinsshowverylittleor
coretranscriptionfactors,suchasTFIIB,TFIIE-aaaa and
no detectable sequence similarity to bacterial transcription
MBF1. MBF1 is the only highly conserved, classical
regulators,theycontaintheHTH structureinthecoreoftheir
HTHdomain thatisverticallyinheritedin allarchaea
DNA-binding domains (9–13). This realization has led to the
and eukaryotes. In contrast, while eukaryotic TFIIB idea that HTH is an ancient DNA-binding module that has
and TFIIE-aaaa possess forms of the HTH domain that beenutilizedforavarietyoftranscriptionregulationprocesses
aredivergentinsequence,theirarchaealcounterparts inthecourseofevolution.
contain typical HTH domains. It is shown that, Subsequent studies on several DNA-binding proteins, such
besidestheHTHdomain,archaeaencodeunexpectedly asthebacterialbiotinoperonregulatorBirA(14)andeukaryotic
large numbers of two other predicted DNA-binding forkhead and H1/H5 (15), have led to the identification of a
modifiedderivativeoftheHTHdomainknownasthewinged-
domains, namely the Arc/MetJ domain and the Zn-
helix domain(wHTH) (16).These proteins, in(cid:2)additiontothe
ribbon. The core transcription regulators in archaea
core three-helixbundle, containa C-terminal -hairpin called
andeukaryotes(TFIIB/TFB,TFIIE-aaaa andMBF1)andin
the wing. In some versions of this domain, the loop betwee(cid:2)n
bacteria(thessss factors)sharenosimilaritybeyondthe
helix 1 (H-1) and helix 2 (H-2) is extended to give rise to -
presence of distinct HTH domains. Thus HTH strandelementsthatinteractwiththetwostrandsofthewing.
domains might have been independently recruited Further sequence–structure analyses have shown that a wide
for a role in transcription regulation in the bacterial rangeofeukaryoticDNA-bindingproteins,suchasHNF3,H1
*Towhomcorrespondenceshouldbeaddressed.Tel:[phone];Fax:[phone];Email:[email protected]
NucleicAcidsResearch,1999,Vol.27,No.23 4659
Table1.FamiliesofarchaealHTHproteins
andHSF,aswellasbacterialtranscriptionregulators,suchas with the bacteria followed by gene displacements. The
ompR,BirAandCAP,assumethewHTHstructure(13,17,18). ‘eukaryotic’genesgenerallyencodethestablecoreofproteins
InadditiontonumerousDNA-bindingproteins,HTHorwHTH involved in translation, replication, repair and transcription,
structures have been detected in RNA-binding proteins, such as whereasmostofthe‘bacterial’genesencodetheevolutionarily
ribosomal protein L11, RNA editing enzymes and some mobile‘shell’components,suchasmetabolicenzymes,structural
aminoacyl-tRNAsynthetases,whichisconsistentwithanancient, proteinsandsignaltransductionmolecules(22,24,25).
generalnucleicacid-bindingfunctionofthesedomains(19–21). Analysis of the readily identifiable components of the
WhilethefunctionsoftheHTHdomaininthecrown-group archaeal transcription machinery revealed a clear eukaryotic
eukaryotes and in bacteria are well understood, there is very affinity.Thecomponentssharedwitheukaryotes,totheexclusion
littleinformationaboutgene-specifictranscriptionalregulators ofbacteria,includetheuniqueRNApolymerasesubunits,the
inthethirddomainoflife,thearchaea.Detailedanalysesofthe TATA-binding protei(cid:3)n, TFIIS, TFIIB (designated TFB in
archaealgenomeshaverevealedtwocategoriesofarchaealgenes, archaea;26)andTFIIE- (Table1;27).Furthermore,phylogenetic
namely those that are most similar to eukaryotic homologs and analysesofthelargeRNApolymerasesubunitsunequivocally
thosethatmostcloselyresemblebacterialhomologs(22,23).Some demonstrated the affiliation between archaea and eukaryotes
uncertaintyduetopossiblelargevariationsin evolutionary rates (28,29). Functional analysis of these subunits and basal tran-
notwithstanding,thissplitmostlikelyreflectsamixedevolutionary scriptionfactorshassuggestedthattheyassembleandfunction
heritageofthearchaearesultingfrommultiplegeneexchanges inaverysimilarwayinarchaeaandeukarya(30).Theseresults
4660 NucleicAcidsResearch,1999,Vol.27,No.23
Table1.Continued
haveledtothegeneralconceptofaeukaryote-typetranscription prompted us to investigate the archaeal transcription
mechanisminthearchaea. machinery, and particularly the transcriptional regulators, in
Detailed comparisons of the first completely sequenced detail.Here,weuseprofilesearchmethodstoidentifytheHTH
archaealgenome,thatofMethanococcusjannaschii,tobacterial proteins from the four completely sequenced euryarchaeal
genomes and a subsequent comparative analysis of the four genomesandclassifythemusingclusteringbysequencesimilarity,
sequenced archaeal genomes have revealed the presence of phylogenetic tree topology and domain architecture. We also
large numbers of HTH proteins that were more similar to investigate the presence of other domains co-occurring with
bacterial HTH/wHTH proteins than to any of their known HTH domains in archaeal proteins and predict the possible
eukaryotic counterparts (22,24,31). Furthermore, preliminary functionsoftherespectiveproteins.Additionally,wedescribe
experimentalcharacterizationofsomeofthesearchaealDNA- other potential transcriptional regulators encoded in the
binding regulators has suggested that they regulate transcription archaeal genomes, such as the Met/Arc transcription factors
similarlytotheirbacterialcounterparts(32,33).Theseobservations (34,35) and Zn-ribbons (36). We discuss the implications of
stood out against the similarity of the core transcription theseobservationsfortheevolutionoftranscriptionregulation
machinery of the archaea to that of the eukaryotes and ingeneral.
NucleicAcidsResearch,1999,Vol.27,No.23 4661
Table1.Continued
aUnlessspecificallyindicatedotherwise,allfamiliesofHTHproteinsshouldbeconsideredarchaea-specific,with
onlyageneric(andnotnecessarilystatisticallysignificantindirectpairwisecomparisons)similaritytobacterial
HTHdomains.Thenumbersinparenthesesindicatethenumberofparalogs(whenevermorethanone)inthegiven
archaealspecies.Speciesnameabbreviations:Af,Archaeoglobusfulgidus;Mj,Methanococcusjannaschii;Mta,Meth-
anobacteriumthermoautotrophicum;Ph,Pyrococcushorikoshii;Aae,Aquifexaeolicus;Hsp,Halobacteriumsp.;Sco,
Sulfolobussolfataricus.
MATERIALSANDMETHODS thatgavemaximumdepthofdetectionandnofalsepositivesin
six iterations were chosen for saving the profile using the –C
Sequences,databasesandsequenceanalysis
option of PSI-BLAST. These profiles were then individually
Thepredictedproteinsequences(proteomes)derivedfromthe run against the four archaeal proteomes using the –R option
four complete euryarchaeal genomes, namely M.jannaschii and preliminary lists of predicted HTH proteins were
(Mj) (37), Methanobacterium thermoautotrophicum (Mta) (38), extracted. These proteins were run against the NR database
Archaeoglobus fulgidus (Af) (39) and Pyrococcus horikoshii using the gapped BLAST program in order to detect other
(Ph)(40),wereobtainedfromthegenomesdivisionofEntrez domainspresentinthemandtoconfirmthepresenceoftheHTH.
and corrected for frameshifts or incorrect starts wherever Asanindependentmodeofdetection,analignmentof the HTH
necessary.Sequenceanalysiswasperformedusingacombina- domainswasextractedfromalibraryofknownbacterialHTH
tion of local alignment searches using the BLAST family of transcription factors using the Gibbs sampling approach as
programsandprofilesearchesusingthePSI-BLAST(41)and implementedinthePROBEprogram(43).Thisalignmentwas
MoST (42) programs. Briefly, the procedure was as follows. used to derive a position-specific weight matrix that was run
Using a variety of known HTH domains as queries, the non- againstthefourindividualarchaealproteomesusingtheMoST
redundant(NR)proteinsequencedatabasewassearchedusing program.Thisapproachwasalsousedastheprincipalmethod
thePSI-BLASTprogramforsixiterations,withthecut-offfor fordetectingtheMetJ/Arcfamilymembersfromarchaealand
inclusionofsequencesintotheprofilesetattheexpectation(e) bacterialgenomes.
valueof0.01.Theoutputswereexaminedforthepresenceof TheHTHproteinswereclassifiedusingtwomethods.(i)Single
false positives, i.e. proteins whose pattern of amino acid linkage clustering as implemented in the Grouper program of
residue conservation and predicted secondary structure were the SEALS package (44). This program uses the bit score of
incompatiblewiththoseseeninHTHdomains.Thesequences thegappedBLASTalignmentasthecriterionforclusteringthe
4662 NucleicAcidsResearch,1999,Vol.27,No.23
proteins.Serialbitscorecut-offsintherange40–75wereused by means of subsequent multiple alignments and secondary
to divide the proteins into clusters of increasing stringency. structure prediction, to represent distinct families of HTH
(ii)In order to delineate orthologous families, the proteins domainsand,accordingly,wereincludedintheheterogeneous
were compared to the NR database and the symmetry of the mHTHcategory.Mostoftheserelationshipswereindependently
besthitsbetweendifferentgenomeswasexamined.Specifically, recognized using multiple alignments constructed with the
if protein X1 from proteome X gave best hits to proteins Y1, Gibbssampling methodimplementedinthe PROBE program
Z1andQ1inproteomesY,ZandQinasearchagainsttheNR astheinputforiterativedatabasesearchingusingMoST.The
database,itwasdeterminedwhetherproteinsY1,Z1andQ1in consistent detection of sequence similarity with different
similarsearchesagainsttheNRgavetheotherthreeproteinsas searchingmethods,togetherwiththedirectevidenceforstructural
thebesthitsfromtheirrespectiveproteomes.Suchadistribution similarity of diverse proteins, strongly suggest that all these
of best hits is termed ‘symmetric’ and is consistent with the HTH domains are homologous. In addition to known HTH
hypothesisthattheseproteinsformafamilyoforthologs. domains and their close homologs, this analysis detected a
Multiple alignments were constructed by combining PSI- number of uncharacterized proteins that can be predicted to
BLAST-generatedpairwisealignmentsandadjustedusingthe adopt the HTH structure, particularly that of the wHTH class
informationonthesecondaryelementsinHTH andArc/MetJ (seebelow).
domains with well-resolved structure, such as LexA, BirA, These searches detected primarily bacterial and archaeal
Cro, c1, CAP and Arc. These alignments were used to build (predicted)HTHdomainsandonlyafeweukaryoticproteins.
phylogenetic trees using the neighbor-joining method as The main classes of detected eukaryotic HTH proteins were
implementedinCLUSTALW/X(45)andthePHYLIPpackage themembersofthePairedandCENP-Bfamilies(13),bothof
(46).Astheinformationinthealignmentcomprisingonlythe whichare distinctand apparentlyrelativelyrecentderivatives
HTH domain was not sufficient to resolve higher order of the transposon integrase HTH domain family (53,54). The
branching, only the terminal branches with bootstrap support POU domain and the homeodomain were detectable with
>70%in500trialswereconsidered. marginalstatisticalsignificance(evalue~0.01)insomeofthe
Secondary structure predictions and structural database searches that were initiated with the transposase-type HTH
threading were performed using the PHD program (47,48). domains (53,55). Thus, with a few exceptions, bacterial and
Protein homology modeling was performed using the archaealHTHdomainsappeartobemuchmorecloselyrelated
PROMOD-IIprogram(49).TheSWISS-PDBviewerwasused toeachotherthaneitherofthemaretotheeukaryoticstructural
to manipulate the pdb files and provide the alignments for counterparts. In searches initiated with eukaryotic queries,
model building. Protein structures were visualized using the such as the homeodomain, myb, H1, forkhead and ETS, the
MOLSCRIPTprogram(50). prokaryoticproteinswerelargelyundetectable,withtheexception
of two Myb-like proteins from Bacillus subtilis (YwfN,
732380; YlbO, 2340012). Having established an apparent
RESULTSANDDISCUSSION affinityofthepredictedarchaealHTHproteinswiththebacterial
ones,weinvestigatedthemingreaterdetail.
Sequence–structure-baseddetectionandclassificationof
As a result of the searches described above, the predicted
HTHdomains
HTH domains were extracted from the four complete sets of
In order to investigate the archaeal HTH domain-containing archaeal proteins. The individual sequences of these domains
proteins in detail, we used structural criteria (see the SCOP wererunagainsttheNRdatabaseusingthesingle-passgapped
database; 51) to divide them into three sets: (i) the(cid:2)winged- BLAST program to identify the closest homologs. To verify
HTH (wHTH) domains that contain the C-terminal -(cid:2)hairpin thepredictionoftheHTHstructure,multiplealignmentswere
and, in some cases, the additional elements of the -sheet made for several of such families and used as queries for
betweenH-1andH-2;(ii)Cro-likeHTH(cHTH)domainsthat secondary structure prediction using the PHD program. In
have no additional elements at the C-terminus and contain a conjunction with the available 3-dimensional structures of
tight loop between H-1 and H-2; (iii) miscellaneous HTH HTH proteins, the resulting predictions were used as a guide
(mHTH), a heterogeneous collection of domains that differ for constructing multiple alignments of all predicted archaeal
from the typical HTH in terms of certain features, such as wHTH,cHTHandmHTHdomains(Fig.S2a–c).
shortened helicesor incorporation into other super-structures, When the alignments of all the different types of the HTH
e.g.thecyclinfoldinTFIIB(52;seeSupplementaryMaterial, domains are superimposed, the sequence signatures underlying
Fig.S1).Foreachoftheseclasses,weinvestigatedtheneigh- the structural conservation become apparent (Fig. S2). Specifi-
bors in protein sequence space using iterative PSI-BLAST cally, H-1 contains a triad of medium sized to bulky residues
searches with the structurally characterized representatives as that typically consists of a polar residue followed by two
starting points (six iterations, e value cut-off of 0.01 for hydrophobic ones (positions 10–12 in Fig. S2). In the cHTH
includingsequencesintheprofile). domains,thedistalpositionofthistriadisinsomecasesoccupied
Using structurally characterized proteins that represent bya basic residue. The mostconservedsignature of the HTH
distinctfamilieswithinthewHTHclassasqueriesforiterative proteins,thedyadofanacidicandahydrophobicresidue(most
searches, we found that all these families recognized each frequently,glutamicacid–isoleucine),islocatedinthemiddle
other;asimilarresultwasseenwiththe cHTH.Inthecaseof of H-2 (Fig. S2, positions 31–32). This signature is followed
the heterogeneousmHTH category, we used separate profiles bythecharacteristicturnwhichissignaledbyapositionatthe
for each family that included the TFIIB/TFB, MerR and end of H-2 that is most often occupied by residues with high
MCM-typefamilies.Someofthesequencesthatweredetected turn-formingpropensity,suchasglycine,asparagine,lysineor
insearcheswithprofilesforthecHTHandwHTHwereshown, aspartate (Fig. S2, position 38). H-3 is the recognition helix
NucleicAcidsResearch,1999,Vol.27,No.23 4663
that makes the major groove contact with DNA and contains
theresiduesthat,inpart,areresponsiblefortheDNA-binding
specificity. This helix contains a highly conserved hydro-
phobicpositioninitsN-terminalpart(Fig.S2,position44)and
abulkypositionintheC-terminalpart.InthewHTHproteins,
the latter position is occupied by a hydrophobic residue,
whereasinthecHTHdomains,itisachargedresidue(Fig.S2,
position51).
Inadditiontotheabovesignaturesthatare seeninallHTH
domains,thereareotherfeaturesthatarerestrictedtospecific
classes. As already mentioned, the wHTH domains show
considerable variability in the regi(cid:2)on between H-1 and H-2,
and in some of them, one or more -strands are found in this
region.Themost(cid:2)characteristic featureofthe wHTHdomains
istheC-terminal -sheet,thewing.ThejunctionbetweenH-3
and the first strand of this sheet is marked by a highly
conservedglycine (position 55 inFig. S2a), whichisa signa-
ture of the wHTH class of HTH domains. While strand 1 is
Figure 1. Relative abundance of HTH, Arc/MetJ and Zn-ribbon domains in
easily discernible and moderately conserved in the wHTH
archaeaandbacteria.Verticalaxis:numberofdetecteddomainsper1000proteins.
sequences,thereisconsiderablevariabilityintheloopbetween
Bacterialspeciesabbreviations:Aae,Aquifexaeolicus;Bs,Bacillussubtilis;
the two strands (Fig. S2a). In the cHTH domains, the region Mtu, Mycobacterium tuberculosis; Ec, Escherichia coli. Note the striking
between H-1 and H-2 does not show much variability but is differences in the distributions of the Arc/MetJ and Zn-ribbon proteins in
characterizedbyaconservedglycinethatmaintainsthestructure contrast to that of the HTH proteins. Also note the low number of HTH
proteinsinMethanobacteriumrelativetotheotherspecies.Thepreliminary
inaclosedconformation(Fig.S2b,position26).Thustheclassical
dataforthecrenarchaeonAeropyrumpernix(Ape)wereincluded(seeAddendum).
cHTH domain is smaller and more compact than the wHTH Thisgenomehasbeenreportedtoencode2694proteins.However,sincethis
domain. These features, in addition to the specificity of the resultsinagenedensitythatismuchlowerthaninotherarchaeaandbacteria
profilesforeachofthesetypesofHTH,helpedindistinguishing andalsobecauseanunusuallylargefractionofthesepredictedproteinshave
nodetectablehomologsinotherspecies(L.AravindandE.V.Koonin,unpublished
these two classes of HTH proteins encoded in the archaeal
observations),weconsiderthistobeagrossoverestimate.Thedatashownin
genomes.InafewofthewHTHdomains,theC-terminalsheet
thefigurearebasedonanestimated1800proteinsencodedintheA.pernixgenome.
appeared to show some signs of degeneration, but their
evolutionaryaffinitiesneverthelesscouldbeestablishedonthe
basis of their specific sequence similarity to other wHTH
proteins.
themarespecialized,conservedversionsoftheHTHdomain,
ThediversityofHTHproteinsinarchaeaandtrendsin such as those seen in the MCM proteins and in TFIIB/TFB
theirevolution (Fig.S2c).
To establish orthologous relationships among the archaeal
Our analysis revealed a much greater diversity of HTH
HTH-containingproteins,weinvestigatedthephyleticdistribution
domains among the archaea than previously suspected, in
terms of both numbers and relationships. The counts of ofthetaxon-specificbesthitswithanevaluethresholdof10–2
detected HTH domains range from 39 in M.thermoauto- for allarchaealHTH domains.Among the214 archaealHTH
trophicum to 88 in A.fulgidus (Fig. 1). Even when the counts domainsthatgavedatabasehitsabovethecut-off,<10%were
werenormalizedbythetotalnumberofgenesinthegenomeof reliably (e value difference of two orders of magnitude or
eacharchaeon,therewasacleardifferenceintheabundanceof greater from the next best hit) most similar to bacterial
theHTHdomains(Fig.1).Archaeoglobusfulgidus,whichhas homologs, whereas for the rest, the best hit was another
the largest genome, encodes more than twice as many HTH archaeal protein. Thus, most of the archaeal HTH domains
domains per 1000 proteins as M.thermoautotrophicum. The appear to have undergone at least some evolution within the
numbers of HTH domains in P.horikoshii and M.jannaschii are archaeal superkingdom. The evolutionary events involved
closertothatinArchaeoglobus,suggestingthatMethanobacterium might have included both vertical inheritance and horizontal
has a deficit of HTH proteins. In each case, the normalized transfer amidst the archaea (24). Those archaeal HTH-
count of HTH proteins in archaea is within the range seen in containingproteinsthatdidgivebesthitstobacterialproteins
free-livingbacteriawithproteomesizescomparabletothoseof mightbecasesofhorizontaltransferfrombacteriaintoarchaea
thearchaeaandonlyslightlylowerthaninbacteriawithlarge (seebelow).
genomes and versatile metabolism, such as B.subtilis and Those archaeal proteins that form symmetrical pairs of
Escherichiacoli(Fig.1). intergenomicbesthitscanbeconsideredlikelyorthologs.This
The wHTH domains are by far the most abundant class of notion is reinforced in cases where a consistent clique of
HTHdomainsinthearchaea(Fig.S2).Therearefiveormore symmetricalbesthitsconnectsthreeorallfourofthearchaeal
times as many wHTH domains than there are Cro-like HTH genomes (24,56). The validity of these putative orthologous
domainsineachofthearchaealspecies;thecountoftheCro-like clusters was additionally evaluated by using single linkage
domainsisaboutthesameineachofthearchaea,namely7–11. clusteringofarchaealHTHdomainsandneighbor-joiningtree
TheremainingtypesofHTHdomains(themiscellaneous class) analysis. Using these procedures, we identified eight clusters
accountforonlyasmallfractionofthetotalnumber;someof oflikelyorthologsthatarepresentinallfourarchaea,15that
4664 NucleicAcidsResearch,1999,Vol.27,No.23
Table2.LikelyoriginofarchaealHTHproteinsbyhorizontalgenetransferfrombacteria
aDatabasehitswithanassociatedevalue<0.01inasinglepass,gappedBLASTsearchareincluded.
bTheC-terminalportionofPH0952thatconsistsofTPRrepeatsandshowssimilaritytoavarietyofproteinswas
removedpriortothedatabasesearch.Databasehitsareindicatedbythegenenamefollowedbyanabbreviated
species name and the gene identification number (in parentheses); the e value is also indicated (e – n = 10–n).
SpeciesnameabbreviationsnotusedinTable1:Av,Azotobactervinelandii;Bs,Bacillussubtilis;Ec,Escherichia
coli;Le,Lycopersiconesculentum(tomato);Rc,Rhodobactercapsulatus;Rl,Rhizobiumleguminosporum.
cDataforMTH659.
are represented in three species and 22 that are found in two Horizontal transfers between archaeal species are hard to
species(Table1). detect due to the general background conservation of protein
Notably, among the eight orthologous clusters of HTH- sequences. In contrast, horizontal transfers from bacteria are
containingproteinsthatareconservedinallfourarchaea,four apparent (Table 2). Clear-cut examples include the BirA-like
have obvious orthologs in eukaryotes. These clusters include proteininArchaeoglobus,theHTH-containing,apoptotic-type
theorth(cid:3)ologsofthebasaltranscriptionfactorsTFIIB(mHTH), (AP) ATPase fro(cid:2)m Pyrococcus and proteins containing the
TFIIE- (w(cid:3)HTH)andMBF1(cHTH)andphenylalanyl-tRNA double-stranded -helix domain in Methanobacterium. A
synthetase -subunit (wHTH). Of the remaining four all- conservative estimate of at least seven cases of horizontal
archaeal clusters, one includes the DtxR-type HTH domains transferofbacterialgenesencodingHTHproteinsintooneof
that have closely related bacterial orthologs involved in iron the four archaeal lineages can be made on the basis of the
metabolism regulation (57), and the other three appear to be BLAST search results, clustering and domain organization of
archaea-specific. the HTH proteins. Additional, ancient horizontal transfers of
Beyondthe smallsetofHTH domainsthatare components bacterialgenesmighthavebeenobscuredbysubsequentinter-
ofthecoretranscriptionmachinery,therestarenotuniversally archaealhorizontaltransfer.Candidatesforsuchancienttransfers
conserved (Table 1). The orthologous sets account for only aretheLysRfamily(9)andtheCadRfamilythatareprevalent
~25% of the total number of HTH-containing proteins in the in bacterial genomes but are represented by only one or two
fourarchaea;thissuggestsprominentrolesforlineage-specific membersintwoofthearchaealgenomes(Tables1and2).
duplication and horizontal transfer in the evolution of the The analysis of orthologous clusters suggests that the
observed diversity. The results of single linkage clustering, commonancestorofthefoureuryarchaealspeciesmostlikely
tree analysis for different HTH classes and the encoded20–30HTH proteins.Thisnumberisconsistentwith
TAX_COLLECTOR analysis indicate that lineage-specific afree-livingprokaryotethat,inadditiontothecoretranscription
gene duplication has been a major force in the evolution of machinery, encoded transcription regulators for some basic
archaeal HTH-containing proteins. These specific expansions metabolic processes that might have included, among others,
are particularly prominent in Archaeoglobus and Pyrococcus ironmetabolism(57).Thesubsequentradiationofthearchaeal
(seebelow). lineages seems to have involved considerable loss of genes
NucleicAcidsResearch,1999,Vol.27,No.23 4665
Figure2.DistinctdomainarchitecturesofarchaealproteinscontainingpredictedHTHdomains.ThediversedomainarchitecturesofthearchaealHTHproteinsare
shownwiththedifferentdomainsdrawntoscale.ThewHTH,cHTHandtheheterogeneousmHTHcategoriesaredistinguishedfromeachother.Theabbreviationsfor
some of the domains shown in the figure are: 4VR, 4-vinyl reductase; Fer, ferredoxin; HAMP, histidine kinase-adenylyl cyclase-methyl accepting protei(cid:1)n-
phosphatasedomain;OPRT,orotatephosphoribosyltransferase;X/GPRT,xanthine-guaninephosphoribosyltransferase;DBHEL,AraC-likedouble-stranded -
helix;Z,Zn-ribbon.Therestofthedomainsaredescribedinthetext;theHINTmoduleisshownassplitINT+EINtoindicatetheinsertionofotherdomainsintoit.
coding for HTH-containing proteins (note the 13 orthologous The rest are small proteins that have no distinct globular
clusters that include three archaeal species in Table 1). This domains other than HTH or contain predicted non-globular
wascompensatedforbyindependent,lineage-specificduplications domains;afewalsocontainpredictedglobulardomainswhose
thatresultedintheaccumulationofcertainfamiliesinparticular identitycouldnotbediscernedbysequenceanalysis.Theaddi-
species,alongwithsomehorizontaltransferfrombacterialand tionalglobulardomainsthatarefusedtotheHTHdomainscan
otherarchaealgenomes.
be classified into enzymatic and interaction-mediating ones.
Figure2showsarepresentativesetofdomainarchitecturesof
Domainarchitecturesandpredictedfunctionsofthe
archaealHTHproteins theseproteinsfromdifferentarchaea.
In several archaeal proteins, a wHTH domain is fused to
AlthoughitisformallypossiblethatsomeofthearchaealHTH
apparentlyactive(asjudgedbytheconservationofallessential
domains are involved in protein–protein interactions rather
catalyticresidues;datanotshown)metabolicenzymes,suchas
thaninnucleicacidbinding,thelatteristhepredominant,ifnot
biotin synthase, orotate phosphoribosyl transferase, xanthine-
the only role of HTH proteins in bacteria. Given the clear
guanine phosphoribosyltransferase and threonine synthase.
relationshipsbetweenbacterialandarchaealHTHdomains,in
terms of sequences, and in many cases, domain organization Theseproteinsmightcombinethe catalyticfunctionwiththat
(see below), the prediction that most of the archaeal HTH of transcription regulation of the biosynthetic genes in
proteinsareDNA-bindingtranscriptionregulatorsseemstobe response to the respective metabolite in the environment.
justified. Severalsimilarcasesareseeninbacteria(13,57,58)butsome
About one-third of the archaeal HTH proteins possess of the combinations of HTH domains with nucleotide
distinctadditionaldomainsthatarefusedtotheHTHdomain. biosynthesisenzymesthusfarappeartobeuniquetothearchaea.
4666 NucleicAcidsResearch,1999,Vol.27,No.23
(cid:2)
Aconservedfamilyofarchaealproteins(e.g.AF1611shown in evolution the double-stranded -helix domain had been
in Fig. 2), which is also represented in thermophilic bacteria, independentlyfusedtoaDNA-bindingHTHdomain.
contains a wHTH domain fused to the N-terminus of a Inaddition tothe proteinsthathaveclear bacterialcounter-
predicted DNA methyltransferase (58). This might be an parts, we detected several archaea-specific fusions of HTH
adaptationofthewHTHfortargetingthemethyltransferaseto domains to small ligand-binding domains (Fig. 2). In the
DNAforthermophile-specificDNAmodification. AF1620 protein, for example, a wHTH domain is combined
The other prominent group of enzymatic domains that are with three PAS domains that are likely to respond to light or
associated with the archaealHTH domainsare ATPases. One redox potential by binding a ligand (64). Several archaeal
suchfusionisseeninthearchaealorthologsofMCMproteins. proteins (e.g. MTH1553 and AF0439) contain fusion of an
MCM proteins play critical roles in the initiation of DNA iron-binding ferredoxin domain to either wHTH or cHTH
replication in eukaryotes (59,60). The archaeal MCM orthologs domains(Fig.2).Giventheimportanceofiron-redoxmetabolism
containadistinctHTHdomainattheC-terminus,whichisnot in thearchaea,itseemspossible thatthe proteinsthatcontain
detectableintheeukaryoticMCMproteins,andeitherrepresents theHTH–ferredoxincombinationfunctionasregulatorsofthe
an archaea-specific fusion or might have been eroded in redox response. Several independ(cid:2)ent fusions of cHTH and
eukaryotes. Perhaps the most notable of the ATPase–HTH wHTHdomainswithcystathionine synthase(CBS)domains,
combinations is the C-terminal fusion with a novel class of which are predicted to function in signal transduction via
archaealATPases(Fig.2).Thisdomainarchitectureisrepresented ligandbinding(65),weredetectedinthearchaea.Thisdomain
inthe genomesof Pyrococcus, MethanococcusandMethano- combinationmightplayaconservedsensorroleinthearchaea
bacterium and also in the incomplete genome of the crenar- since the cHTH–CBS architecture is conserved not only in
chaeon Sulfolobus sulfotaricus, suggesting a widespread three of the four completely sequenced genomes but also in
distribution in the archaea. These ATPases are particularly S.sulfotaricus.Similarly,theaspartokinase-chorismatemutase-
abundantinPyrococcus,whichencodesatleasteightmembers TyrR (ACT) domain (66), a predicted amino acid-binding
of this family (24). The combination of the ATPase domain domain involved in allosteric regulation of a number of
with the HTH domain suggests that these proteins might enzymes, is fused to a HTH in the MJ0241 protein. The
possess a helicase-like activity and play a regulatory role in tendency of HTH domains to combine with various small
DNA unwinding or might be involved in reorganizing DNA- ligand-bindingdomainsaswellasmetabolicenzymessuggests
associatedproteincomplexes. thatinarchaea,transcriptionrespondstotheenvironmentina
Another remarkable domain architecture is the fusion of an mannerakintothatinbacteria.
HTHdomaintotheN-terminusofapredictedS/T-typeprotein AninterestingobservationisthedetectionofcHTHdomains
kinase domain of the Rio1 family (61) that is conserved in in a number of archaeal inteins (e.g. those in the replication
A.fulgidus, P.horikoshii and M.jannaschii. An orthologous factorCandinitiationfactor2fromM.jannaschii).TheseHTH
proteinwiththesamedomainarchitecturewasalsodetectedin domains are sandwiched between the N-terminal part of the
eukaryotes (yeast YNL207w). This unusual protein kinase Hedgehog–intein (HINT) module and the inserted homing
might be an ancient regulatory protein that could function endonucleasedomain(67,68;Fig.2).Thisassociationwiththe
through phosphorylation of DNA-associated protein endonucleasedomainsuggeststhattheHTHmightplayarole
complexes. in the recognition of target sequences by the endonuclease in
The fusion of the wHTH dom(cid:3)ain to the N-terminus of the theprocessofhoming.
phenylalanyl-tRNA synthetase -subunit is a relatively rare SomearchaealHTHproteinsalsocontainanotherpredicted
casewhenanHTHdomainseemstobeusedforRNA-binding DNA-binding domain, na(cid:3)mely the Zn-ribbon. The core tran-
rather than transcriptional regulation. The wHTH domain in scription factors TFIIE- and TFB are combinations of
these proteins is similar to those seen in other RNA-binding different forms of the HTH domain with the Zn-ribbon. The
proteins,suchastheRNA-editingdeaminaseandtheeukaryotic archaealorthologsoftheeukaryoticMBF1protein(69,70)also
ribosomalproteinS10(21). combine a cHTH with a Zn-ribbon which is absent in their
HTH domains in the archaea are also fused to a variety of eukaryoticcounterparts.
non-enzymaticdomainsthatarepredictedtomediateinteractions Auniquefamilyofarchaea-specificHTHproteinsisdefined
with other proteins, nucleic acids or small molecule ligands byMJ1243anditsorthologsandisrepresentedbyonemember
(Fig. 2). In bacteria, HTH domains typically are fused to in all four archaeal genomes. These HTH proteins contain a
domainsrecognizingsmallmoleculesthatregulatetheexpression basic patch that is located N-terminally of the HTH and is
of various operons. Homologs of some of these proteins with predicted to adopt an AT-hook-like minor groove-binding
similar domain com(cid:3)binations were detected in the archaea. surface (71). This combination of major and minor groove-
These in(cid:2)clude the -helical iron-binding domain and the C- bindingmotifsechoesthesynergismbetweentheAT-hookand
terminal -barreldomainpresentinthe(cid:2)DtxR-likeproteins(57) otherDNA-bindingdomainsineukaryotes(71).
(e.g. MJ0568 and MTH936) and the -jelly roll domain (62)
Othernucleicacid-bindingproteinswithapossiblerolein
thatisfusedtoLysR-likeHTH domains(AF2127,MTH1545
archaealtranscription
and MJ1120). Methanobacterium thermoautotrophicum
contains two proteins (MTH659 and MTH700) with Cro-like TheArc/MetJfamilyoftranscriptionfactors.WhileHTHisthe
HTH dom(cid:2)ains that are fused to the N-terminus of a double- predominant DNA-binding domain in prokaryotes, several
stranded -helix domain similar to those found in vicilin and otherunrelatedtranscriptionfactorfamilieshavebeenfoundin
phaseolin and probably involved in sugar recognition (63). A bacteria. One of these inc(cid:2)ludes the MetJ/Arc proteins that act
similar domain is fused to the N-terminus of the wHTH in byinsertinganN-terminal -strandintothemajorgrooveofDNA
AraC-type transcription factors, suggesting that at least twice (34,72). In addition to this strand, they possess two C-terminal
NucleicAcidsResearch,1999,Vol.27,No.23 4667
heliceswhoseorientationisreminiscentofthetwoC-terminal the cooperation between the Arc/MetJ and PIN domains in
helicesofHTHproteins(73).However,ratherthanparticipating transcriptionregulationmightbeageneralphenomenon.
in DNA binding as they do in the classical HTH domains, in
the MetJ/Arc family proteins, these helices are involved in Therubredoxin-likeZn-ribbons.Therubredoxin-likeZn-ribbonis
dimerizationof the domain (Fig.S3a). Mostof these proteins a small metal-binding module that is widespread in proteins
areverysmallanddivergentinsequence,althoughtheyseem fromallthreedomainsoflife(51,75).Zn-ribbonsparticipatein
toadoptaconservedtertiarystructure;thismakesitdifficultto many different functions that include: (i) DNA binding, e.g.
identifythembysequencecomparisonalone. the C-terminal domains of topoisomerase I, TFIIS and RNA
To investigate this family as completely as possible, we polymerase subunits (36); (ii) RNA binding, e.g. ribosomal
performediterativePSI-BLASTsearchesusingallknownArc/ proteinsS27,L37,L40andaminoacyl-tRNAsynthetases(74);
MetJproteinsasqueriesandretrievedallhomologsdetectable (iii)redoxreactions,e.g.rubredoxins,rubrerythrinsandnigre-
by thisapproach. Analignmentofthe DNA-bindingdomains rythrins (75); (iv) Zn-ribbons found as inserts within other
from all these proteins was constructed using the PROBE domains where they probably play a strucuturalrole or aid in
program. Even more divergent members were then detected catalysis,asinthecaseofadenylatekinase(78)andpyruvate
using the MoST program to iteratively search the database formate lyase. The structure of this domain is typified by
withaweightmatrixderivedfromaconservedungappedblock rubredoxinfromDesulfovibriogigas(79)andhasasitscorea
extractedfromthealignment.Thealignmentwassubsequently Zn2+ioncoordinatedbytwopairsofcystein(cid:2)es,eachassociated
refined using secondary structure prediction. PDB database withamoreorlesssymmetricpairofshort -strands(Fig.S4).
threadingusingthePHDprogramwithmultiplealignmentsas Beyond the four strands that surround the bound Zn2+ ion,
input provided strong support for these proteins adopting the differentmembersofthisfamilyshowconsiderablestructural
Arc/MetJ fold. Specifically, the Arc/MetJ structures consist- elaborationintheformofinsertionsbetweenthetwocysteine-
entlyfiguredasthebesthitswithZscoresintherange2.1–2.8. containing pairs of strands. This makes the spacing between
Examinationofthemultiplealignmentandcomparisonwith thecysteinepairsextremelyvariable.
the3-dimensionalstructureoftheArcproteinfromphageP22 Due to this variability, comprehensive detection of Zn-ribbon
shows that the conservation pattern of this family correlates domainsbasedonsequencealoneisparticularlychallenging.We
withbothDNAinteractionsandstructure-stabilizingpropertiesof attemptedtoovercomethisdifficulty,atleastinpart,byusing
the residues (Fig. S3b). In both the first strand and the two transitivesearcheswithPSI-BLASTfollowedbyevaluationof
followinghelices,asignificantpartoftheconservationrestson theconservedresiduepatternswithrespecttotheircompatibility
hydrophobicresidueswhosesidechainspointtotheinteriorof withtheZn-ribbonstructure.Additionally,theconservationof
thestructureandstabilizeitviahydrophobicinteractions.The thecysteinesmakesitpossibletoapplypatternsearchesforthe
conserved hydrophobic residues in H-2 are likely to stabilize detectionofZn-ribbons.Giventhemultiplefunctionsofthese
inter-monomercontacts.Theconservationofalltheseresidues modules,thepresenceofaZn-ribbon,assuch,isnotindicative
in the Arc/MetJ superfamily is consistent with these proteins ofDNAbindingorafunctionintranscriptionregulationforthe
adopting a similar fold and dimeric organization. Another set respective protein. However, given that they are common in
of conserved residues have small side chains that favor the transcription factors and RNA polymerase subunits from
turn-like conformationbetweenstrand-1 andH-1andthetwo archaea, eukaryotes and bacteria, we systematically surveyed
C-terminalhelices.Mostoftheremainingconservedpolarresi- the archaeal genomes for Zn-ribbons. This analysis revealed
dues are in positions that are compatible with DNA interaction. remarkable abundance of Zn-ribbon proteins in archaeal
Thesepositionsincludetheresidueinstrand-1thatisinserted proteomes which, when normalized for the gene number,
into the major groove of DNA and forms hydrogen bond exceededthenumberofsuchproteinsinbacterialgenomesby
contacts with the bases. Thispolar position is likely to playa atleastafactorof2(Fig.1).Basedonsequenceconservation
roleinDNA-bindingspecificityoftheseproteins(Fig.S3aand among Zn-ribbons themselves and the presence of additional
b), along with the non-conserved part of strand-1. The other domains, weclassifiedthe Zn-ribbon-containingproteinsinto
polarresidues,suchastheoneatthebeginningofH-1andthe groupsthatarelikelytorepresenttheirfunctionsintranscription,
oneaftertheturnbetweenthe twohelices(Fig. S3)are likely DNAreplicationandrepair,translationorRNAbinding,redox
toinfluencetheinteractionswiththebackboneoftheDNA. processes and other miscellaneous processes (Fig. S4). This
Our analysis showed that Arc/MetJ proteins are far more groupingshowsthatasignificantnumberofarchaealproteins
common in both archaea and bacteria than previously thatcontainZn-ribbonsarelikelytobeinvolvedinnucleicacid
suspected(Fig.1).Theseproteinsweredetectedinallarchaea, bindingand,morespecifically,intranscription(TableS1).
including Sulfolobus, with the greatest number (14) found in The conserved archaeal Zn-ribbon proteins that have well-
A.fulgidus. This suggests that a distinct family of Arc/MetJ-like understood functions in transcription are compone(cid:3)nts of the
DNA-binding proteins was already present in the common core machinery that include TFIIB/TFB(cid:4),(cid:4) TFIIE- (cid:4), TFIIS/
ancestor of the archaea. We found that this family is nearly RPOM(tworepeats),MBF1andtheRPOE andRPB subunits
ubiquitous in bacteria also, with the maximum number (20 of RNA polymerase. In addition to these core transcription
proteins)presentin Mycobacterium.Plasmid-encoded Arc-MetJ components, the Zn-ribbon is fused to a number of HTH
familyproteinsappeartoregulatethetranscriptionofplasmid domains,andtherespectiveproteinsarepredictedtofunction
genesbyinteractingwithplasmid-encodedproteinsofanother astranscriptionregulators(Fig.2).SomeconservedZn-ribbon
family (74) that we have shown to contain the PIN (PilT N- proteinsthataresimilartothesmallRNApolymerasesubunits
terminal) domain (24). The PIN domain family is greatly andaresharedbyarchaeaandeukaryotes,suchastheMJ1474
expanded in Archaeoglobus and Mycobacterium, which also andMJ0890families(Fig.S4andTableS1),mayrepresentyet
encodethemaximumnumberofArc/MetJ-likeproteins.Thus, uncharacterizedcoretranscriptionfactors.Examinationofthe