W350–W353 Nucleic Acids Research, 2007, Vol. 35, Web Server issue doi:10.1093/nar/gkm393 TFAM 1.0: an online tRNA function classifier Helena Ta˚ quist, Yuanyuan Cui and David H. Ardell* Linnaeus Centre for Bioinformatics, Uppsala University, Box 598, SE-751 24 Uppsala, Sweden Received January 31, 2007; Revised April 3, 2007; Accepted May 1, 2007 ABSTRACT We have earlier published an automated statistical classifier of tRNA function called TFAM. Unlike tRNA gene-finders, TFAM uses information from the total sequences of tRNAs and not just their anticodons to predict their function. Therefore TFAM has an advantage in predicting initiator tRNAs, the amino acid charging identity of nonstandard tRNAs such as suppressors, and the former identity of pseudo- tRNAs. In addition, TFAM predictions are robust to sequencing errors and useful for the statistical analysis of tRNA sequence, function and evolution. Earlier versions of TFAM required a complicated installation and running procedure, and only bacter- ial tRNA identity models were provided. Here we describe a new version of TFAM with both a Web Server interface and simplified standalone installa- tion. New TFAM models are available including a proteobacterial model for the bacterial lysylated isoleucine tRNAs, making it now possible for TFAM to correctly classify all tRNA genes for some bacterial taxa. First-draft eukaryotic and archaeal models are also provided making initiator tRNA prediction easily accessible genes to any researcher or genome sequencing effort. The TFAM Web Server is available at http://tfam.lcb.uu.se INTRODUCTION The vast majority of new tRNA gene sequence data accumulates today from analysis of genome sequences. The major tRNA gene-ﬁnders in use today, tRNAscan-SE (1) and ARAGORN (2), classify the functions of predicted tRNA genes by structurally locating and decoding their inferred anticodons according to an assumed genetic code. As a result, genome projects regularly misclassify initiator tRNAs in genomes from all three phylogenetic domains and lysylated isoleucine tRNA (kIle) genes from bacteria (described further below). These two types of tRNAs carry the same genetically templated anticodons as methionine elongators and hence cannot be distinguished from them by anticodon-based tRNA classiﬁers. It may also happen that these genes are entirely missing in the annotation of a complete genome. Because genome projects regularly verify the completion of their assemblies by checking for the presence of a complete set of tRNA gene classes in their genome data, the ability to identify these two additional classes of tRNA genes provides additional power for this important task [although in very rare cases the lysylated isoacceptor may be missing along with corresponding metabolic pathways (3)]. Furthermore, a method that uses entire sequence information to classify tRNA gene function is more robust to sequencing error, can correctly predict tRNA charging speciﬁcity in organ- isms with altered genetic codes, can predict the identity of suppressors, and can predict the potential or former charging identity of tRNA-like molecules and pseudo- tRNAs (in this article we loosely use ‘‘tRNA’’ to mean both tRNA and tRNA gene sequences). We present here a new version (1.0) of such a method— an update to the TFAM statistical classiﬁer of tRNA function published earlier (4). TFAM 1.0 is now available online as a Web Server requiring no installation. Version 1.0 of TFAM also provides eukaryotic and archaeal tRNA functional models for the purpose of identifying initiator tRNAs, and an expanded bacterial model that can predict some kIle tRNAs. For certain bacterial species such as proteobacteria and gram-positive bacteria, TFAM 1.0 can be expected to correctly annotate the function of all tRNAs with good conﬁdence. In bacteria, the cytidine in the CAU anticodons of lysylated isoleucine tRNAs are post-transcriptionally modiﬁed to lysidine (5). This modiﬁcation simultaneously changes the codon reading speciﬁcity of this usually minor isoacceptor from AUG to AUA and its amino acid charging speciﬁcity from methionine to isoleucine in keeping with the genetic code. Because the unmodiﬁed (CAU) tRNA is charged with methionine (6) it cannot be expected that TFAM would recognize this tRNA as an isoleucine tRNA. However, the determinants that target this tRNA for post-transcriptional modiﬁcation are them- selves genetically templated in the tRNA gene (7,8), and it is possible for TFAM to distinguish this class of tRNA with fairly good conﬁdence when suitably trained (3). Nonetheless, both experimental (8) and bioinformatic (3) evidence suggest that the determinants for this class have diverged in bacteria. Rather than iteratively accreting all *To whom correspondence should be addressed. Tel: +46 18 471 66 94; Fax: +46 18 471 66 98; Email: david.ardell@lcb.uu.se. 2007 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.