Hidden patterns abound in genome sequences. Sophisticated mathematical algorithms spot them. As of now, several powerful tools
exist for identification of transfer-RNA genes from genomes. These sometimes fail to identify when introns are at noncanonical sites. We discuss our
approach to this problem of identification and apply it to the genome of Nanoarchaeum equitans. Using our algorithm, we identify the four tRNA
genes that were missed by the present standard tRNA search programs in N. equitans. The recent split-tRNA hypothesis [Nature 433, 537 (2005)]
identified the missing ones. However, our solutions are different. We argue the case in favour of our solutions.Author Affiliation: Jayprokas Chakrabarti, Satyabrata Sahoo, Bibekanand Mallick, Smarajit Das and Zhumur Ghosh
1.Computational Biology Group (CBG), Department of Theoretical Physics,
Indian Association for the Cultivation of Science, Kolkata-700 032, India
E-mail : tpjc@iacs.res.inComputational Biology Group (CBG), Department of Theoretical Physics,
Indian Association for the Cultivation of Science, Kolkata-700 032, Indi

Chakrabarti, Jayprokas

Das, Smarajit

Ghosh, Zhumur

Mallick, Bibekanand

Sahoo, Satyabrata

IACS Institutional Repository

Indian J. Phys. 79(6), 559-562  (2005)© 2005 IACS*Corresponding AuthorRapid CommunicationANINTERNATIONAL JOURNALIJPAlgorithm for pattern recognition in nano-sized archaeaJayprokas Chakrabarti*, Satyabrata Sahoo, Bibekanand Mallick,  Smarajit Das and Zhumur GhoshComputational Biology Group (CBG), Department of Theoretical Physics,Indian Association for the Cultivation of Science, Kolkata-700 032, IndiaE-mail : tpjc@iacs.res.inReceived 1 April 2005, accepted 29 April 2005Abstract : Hidden patterns abound in genome sequences. Sophisticated mathematical algorithms spot them. As of now, several powerful toolsexist for identification of transfer-RNA genes from genomes. These sometimes fail to identify when introns are at noncanonical sites. We discuss ourapproach to this problem of identification and apply it to the genome of Nanoarchaeum equitans. Using our algorithm, we identify the four tRNAgenes that were missed by the present standard tRNA search programs in N. equitans. The recent split-tRNA hypothesis [Nature 433, 537 (2005)]identified the missing ones. However, our solutions are different. We argue the case in favour of our solutions.Keywords : tRNA, split-tRNA hypothesis, tRNA search program, Nanoarchaeum equitans.PACS Nos. : 87.14.-g, 87.15.-v, 89.20.-a, 89.75.-k1.  IntroductionMany sophisticated nonlinear algorithms [1] exist forpattern formation and recognition [2,3]. For instance,there are several computational approaches to detecttransfer-RNA (tRNA) genes from a genome [4]. ThesetRNA genes have characteristic pattern over the genomes.To identify these on the sequences, there are algorithms.Notable amongst these are tRNAScan-SE [5] andARAGORN [6]. Most of these tRNA search programskey on primary sequence patterns and/or secondarystructures specific to tRNAs. Quite a few loopholes exist.These have to do with the inability of existing routinesto identify tRNA genes with noncanonical introns inthem. These are unusually located introns in tRNA genes(tDNAs). The standard (canonical) introns are locatedbetween bases 37 and 38 in tDNA. The noncanonicalintrons are the ones located elsewhere [7]. Identificationof tDNAs harbouring these noncanonical introns is thesubject of this paper. Some of the tRNA genes are eithermisidentified or missed by existing search algorithms. Inthis work, we discuss some of these misidentified andnon-identified tRNA genes in the nano-sizedNanoarchaeum equitans Kin4-M (N. equitans for short)by our in-house algorithm.N.equitans (NC_005213) belonging to the novelarchaeal phylum ‘Nanoarchaeota’[8], so far seem to havethe smallest genome of all known cellular life forms.N. equitans is a hyperthermophile. This is the mostcompact, with 95% of the DNA predicted to encodeproteins or stable RNAs. It is the smallest genomeresembling an intermediate between smallest livingorganism like Mycoplasma genitalium  and big viruseslike pox virus. Many symbiotic or parasitic bacteria havesmall cells and reduced genomes but within archaeaN. equitans is the first reported archaea to have suchcharacteristics. Again, its extreme living conditionscorrelate to early environmental conditions suggestingthat ‘Nanoarchaeota’ are a primitive form of microbiallife.The primary tRNA sequence changes to secondarycloverleaf structure [9]. The secondary structure of tRNAhas : (i) Acceptor or A-arm. In this, 5' and 3' ends oftRNA are base paired into a stem of 7 bps (ii) DHU orD-Arm. Structurally a stem-loop, D-Arm frequently560 Jayprokas Chakrabarti et alcontains the modified base dihydrouracil. (iii) Anticodonor AC-arm, made of a stem and a loop containing theanticodon. The canonical structure of AC-loop is essentialfor interactions with ribosomal A and P sites duringprotein synthesis [10]. At 5' end of this loop is apyrimidine base at 32, followed by an invariant U at 33.The anticodon triplet, at 34, 35, 36 is in the exposedloop region. (iv) An Extra Arm, or V-Arm. This arm isnot always present. It is of variable length and is largelyresponsible for the variation in length of tRNAs. Theclassification of tRNAs into types I and II, depends onlength of V-arm [11]. (v) T-y-C Arm or T-arm : Thisarm has conserved sequence of three ribonucleotides :ribothymidine, pseudouridine and cytosine. T-arm hasstem-loop secondary structure and (vi) tRNA terminateswith CCA at 3' end. In case CCA is absent in tDNA, itis added during maturation to tRNA.The attachment of amino acid to their correspondingtRNA is catalyzed by aminoacyl-tRNA synthetase (AARS)[12,13]. Accurate acylation of tRNA depends on twofactors : a set of nucleotides in tRNA molecule (identityelements) responsible for proper identification by AARS[14] and competition between different synthetases fortRNAs [15]. Tertiary L-shape of tRNA facilitates itsidentification by AARS for aminoacylation. L-shape comesabout through the interaction between D-arm and T-arm.There are a few key features that maintain the L-shapeof tRNA [16]. These interactions include Watson-Crickbase pairing, Hoogsteen base pairing, and triple-helicalbase pairing. It is generally accepted that the majorinteractions maintaining the L-shape occur at the cornerof the molecule where D- and T-loops meet. This region,called DT [17], contains several elements, including thereverse-Hoogsteen bp U54:A58 and C55-mediatedU-turn in T-loop, the inter-loop bps G18:C55 andG19:C56 and stack of four mutually intercalated purinebases A58–G18–R57–G19. This intra-loop U54:A58 isstacked on G53.C61 at the end of T stem and forces thetwo bases at positions 59 and 60 to loop out, forming acharacteristic T-loop of 5 bases instead of 7. Thischaracteristic T loop conformation is important forrecognition by elongation factors.The genome of N. equitans consists of a single,circular chromosome of 490, 885 base pairs (bp). It hasan average G+C content of 31.6% [18]. Presumablybecause of this small genome, this archaea has anunusually high gene density, and stable RNA sequences,together covering 95% of the genome. 38 tRNA genesare reported and cross-checked using standard routines(tRNAScan-SE and ARAGORN) include an unusualsecond copy of tRNASer(CGA). However, four tRNAgenes (for glutamate, histidine, tryptophan and initiatormethionine) remained unidentified in the genome. This isdue to their unusual sequence or structure. We identifythem now using our in-house-developed software.Recently, these missing tRNAs were identified using anew split-tRNA hypothesis. However, our solutions aredifferent. We argue the case in favour of our solutions.2.  MethodologyThe entire genome is obtained from NCBI (http://www.ncbi.nlm.nih.gov), accession no. NC_005213. RawtDNA sequences are found by searching the differentmotifs present in the consensus sequence of differenttDNAs of archaea. At first, we adopted the standardcloverleaf model for studying the secondary structure ofpredicted tDNAs of N. equitans. In doing so, we gotsome false positives and a few tDNAs were missed out.We then imposed constraints, unique to archaeal tDNA.A regular cloverleaf structure was searched in tRNAgenes of the genome of N. equitans by adopting archaealtDNA features. The constraints of lengths of stems ofregular tDNA, acceptor arm, D-arm, anticodon arm andT-arm are 7, 4, 5 and 5bp respectively. That asideparameters and constraints used in the search for cloverleaftDNAs are : (a) T8 (except Y8 in M. kandleri), G18,R19, R53, Y55, and A58 are considered as conservedbases for archaea. (b) the lengths of introns and V-armare allowed from 6 to 121 and up to 21 respectively; (c)positions optionally occupied in D-loop are 17, 17a, 20aand 20b; (d) canonical and noncanonical introns may ormay not be present. Keeping these constraints, we wereable to extract 38 tDNAs. After getting the tDNAs, weran the standard routines to check for the secondarystructure. We developed consensus tRNA sequences forarchaea and measured homology with tRNA of N. equitansas a further check.3.  Results and discussionThe recent algorithm [19] for five split tDNAs inN. equitans is new. It locates missing tRNATrp, tRNAiMet,tRNAGlu and tRNAHis. But the split tRNATrp(CCA) solutionis anomalous; the tRNAiMet solution [19] lacks cognitionelements for aminoacylation. In view therefore, we presenthere alternate non-split composite solutions for tRNATrp,tRNA iMet, tRNAGlu and tRNAHis.Earlier [8], tRNA genes in N. equitans wereexhaustively explored. The remarkable algorithms561Algorithm for pattern recognition in nano-sized archaeatRNAScan-SE [5] and ARAGORN [6] located all tRNAsexcept tRNATrp, tRNAiMet, tRNAGlu and tRNAHis. Thenew algorithm of Randau et al [19] locates these missingones. However, the tRNATrp(CCA)  reported [19] isanomalous :  (i) There  is GG preceding the anticodon.We studied all archaeal tRNATrp(CCA) and found this tobe an exception. U33 is known [20] to contribute totRNA-ribosomal binding. Its absence is puzzling. (ii)Further, archaeal tRNATrp(CCA) always have  discriminatorbase A73. This discriminator A73 is of modest preferencefor aminoacylation [21].  Randau et al [19] have C73.Again, the 73rd discriminator base of archaealtRNAiMet(CAU) is always A73. But, tRNAiMet(CAU)solution [19] is anomalous, it has U73.In the absence of conclusive aminoacylation experimentand the anomalies listed above, we reanalyzed the missingtRNAs for Nanoarchaea. In the split-tRNA hypothesis[19], the structures (5-primed end split at 37 followed byinvert-repeat element, 3-primed end preceeded by invert-repeat element etc.) of  tDNA-Glu/His are similar totDNA-Trp/iMet.  If tRNA-Trp/iMet are anomalous, howfunctional are tRNA-Glu/His? Are there other solutions?From the classic work [22] (and the references therein)on tRNA, it is known that archaeal tRNA harbournoncanonical introns. Canonical introns are locatedbetween bases 37 and 38 of tRNA; noncanonical intronsoccur elsewhere. We looked for the possibility that tDNA-Trp/iMet/Glu/His have noncanonical introns. We foundcomposite solutions that do not suffer from the anomaliesabove. These solutions are :Here, we have marked tRNAs in bold italics, intronsin normal font within the gene sequence, the conservedarchaeal Box A promoter-elements [23] in larger fontpresent ahead of the gene. We found the right secondarystructures for all these tRNAs, and the bulge-helix-bulge(BHB) motifs. Note, for instance, the following importantfeatures of this tRNATrp(CCA) : U8, A14, A21, U33,G18:U55, G19:C56, U54:A58 and G30:C40, the anticodonCCA at 34, 35, 36, and finally A73. These bases/base-pairs are conserved in all tRNATrp(CCA) in archaea.tRNAScan-SE identifies bases 151992 to 152081 astRNASer(CGA). Note there is another tRNASer(CGA)between 486337 and 486426.  The one between 151992and 152081 is unlikely to be tRNASer(CGA) : none ofthe conserved bases/base pairs of archaeal tRNASer viz.G1:C72, G18:U55, G19:C56, U54:A58, G26:U44,G53:U61, U33, G73 appear. Again, the Variable-arm isabsent. It is known [24] that G73 and Variable-armcontain identity elements for Ser-RS.From our study of 22 fully sequenced archaea, the73rd discriminator base of tRNAiMet(CAU) is A73. OurtRNAiMet(CAU) has A73. It shares all features of archaealtRNAiMet(CAU).Remarkably, our tRNAGlu(CUC) and tRNATrp(CCA)overlap with one another. Note that the tDNAGlu(CUC)has a noncanonical intron at 33. tDNATrp(CCA) has anoncanonical intron at 30.  N. equitans has the smallestgenome known. Noncanonical introns here compactifytwo tDNAs. Interestingly, this compactification is at workfor tRNAHis as well.Codon usage study of histidine in 22 archaea revealsthe ratio of the number of CAU-codon to CAC-codon tobe anomalously high in N.equitans. Amongst archaea N.equitans is special in this respect. For tRNAHis ATG isthe likely anticodon. This is precisely what we found :tDNAHis(ATG)  lying between 327362 and 327520. It hastwo noncanonical introns located between 32/33 and 71/72 of 13 and 25 bases respectively. In addition to these,there is the canonical intron of 53 bases. Remarkablyagain, this tDNAHis(ATG)  overlaps with tDNAeMet(CAU),located between 327362 and 327500. tDNAeMet(CAT) hasa canonical intron of 66 bases.Randau et al’s split-tRNA solutions are new. Splittingdecompactifies the genome. Further, some of the splitsolutions are anomalous. Our solutions have overlappingcomposite tRNA genes [25]. tRNA genes are woventogether by introns. 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Algorithm for pattern recognition in nano-sized archaea

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