over the past few years , people have witnessed an exponential growth in the number of completed microbial genomes . it is imperative to annotate a genome as precisely as possible , especially due to flourishing with genome - based experimental approaches such as dna microarrays and protein arrays ( 1 ) . specifically , accurate translation initiation site ( tis ) annotation is important for experiments such as identifying native purified proteins through n - terminal amino acid sequencing , as well as heterologous protein products ( 1 ) . meanwhile , in silico studies of translation initiation mechanism , gene regulation , as well as the predictions of operon , promoter and small - untranslated rnas also rely on the correct tis annotation ( 2,3 ) . a tis can be reliably identified by means of the experiments such as n - terminal protein sequencing . unfortunately , such data constitutes only a small portion of all known proteins . even for the best - studied genome escherichia coli k-12 , as collected in ecogene ( 4 ) , less than a quarter of proteins have been verified in such way . nevertheless , as the number of proteomic projects increases , the amount of tis with experimental evidences is expected to accumulate significantly in the near future ( 5 ) . on the other hand , progress has been made in reliable tis identification through computational evidences such as sequence alignment ( 2,6 ) . ( 6 ) was the first to annotate open reading frames ( orfs ) that have significant matches to known proteins with hits distributed in a way to ensure the 5 most candidate start codon to be true . recently , makita et al . ( 2 ) introduced another method to identify high - quality tis by sequence alignment between orthologous genes and applied the resultant dataset to evaluate the performance of tis prediction . the sequence patterns around tiss have been frequently used for in silico study of translation initiation mechanism , thus to design tis prediction algorithm ( 2,712 ) . in textbooks , ribosome is recruited to mrna to initiate translation by specific signals nearby tis such as start codon and shine - dalgarno ( sd ) signal ( 13 ) . however , for genes without ( or almost no ) 5utr in the mrna , i.e. leaderless genes , transcriptional signal such as pribnow box ( bacteria ) or tata box ( archaea ) instead of the sd signal has been found upstream of the tis ( 9,1416 ) . recently , a comprehensive study on hundreds of prokaryotic genomes revealed that  non - sd - led genes are as common as sd - led genes ( 17 ) . thus , it is reasonable to expect that the complexity of prokaryotic translation initiation mechanism will attract a closer attention as more and more genomes are being sequenced . in addition to experimental data in the public database such as swiss - prot , the increasing amount of sequenced genomes , which covers a wide range of prokaryotic branches , allows now a high - throughput approach to systematically collect confirmed tis through database scanning and sequence alignment . it is also interesting to combine the state - of - the - art prediction tools to refine the current public database annotation . moreover , annotating regulatory signals upstream of tis will facilitate the studies of initiation mechanism . herein , we describe a comprehensive relational database , protisa , which is designed to collect confirmed tis , as well as to annotate potential transcriptional or translational signals adjacently upstream to tiss for each of the current hundreds of prokaryotic genomes . we expect that the database may serve the prokaryotic genome annotation and facilitate a wide range of studies on translation initiation . confirmed tiss ( ipt , cdc and hsc ) were collected through database scanning , literature survey , conserved domain search and sequence alignment as follows : 
 imported ( ipt ) : a script was written to extract high - quality manual annotation in swiss - prot . init_met is used to indicate whether the initiator methionine has been cleaved off or not . finally , the ipt data set has been enriched by a simple n - terminal sequence comparison between closely related species.conserved domain confirmed ( cdc ) : the method to identify tis through conserved domain search is essentially similar to that in ( 6 ) . the tis for a gene with only one possible start codon upstream to the 5-most conserved domain hit was readily identified . to compensate for random matches , which would perhaps lead to an incorrect cdc - tis annotation , we removed six amino acids from the most upstream hit before processing ( 18 ) , since the frequency that a hit overlaps with the non - coding region by more than six amino acids is < 1% when examined on genes with ipt tiss.high similarity confirmed ( hsc ) : identifying tis through sequence alignment between orthologous genes has been described in ( 2 ) . briefly , it determines the tis of a gene by referring to its orthologous gene with known tis from other genera . it requires that they are aligned in the n - terminal region . in protisa , genes with tis labeled as ipt or cdc constitute the references to determine hsc tiss . we have been aware that errors in the ipt or cdc tiss might propagate into the hsc tiss via sequence alignment , especially among closely related genomes . to minimize such errors , a hsc tis is annotated only if it has support of orthologous genes from more than one different genus . imported ( ipt ) : a script was written to extract high - quality manual annotation in swiss - prot . init_met is used to indicate whether the initiator methionine has been cleaved off or not . finally , the ipt data set has been enriched by a simple n - terminal sequence comparison between closely related species . conserved domain confirmed ( cdc ) : the method to identify tis through conserved domain search is essentially similar to that in ( 6 ) . the tis for a gene with only one possible start codon upstream to the 5-most conserved domain hit was readily identified . to compensate for random matches , which would perhaps lead to an incorrect cdc - tis annotation , we removed six amino acids from the most upstream hit before processing ( 18 ) , since the frequency that a hit overlaps with the non - coding region by more than six amino acids is < 1% when examined on genes with ipt tiss . high similarity confirmed ( hsc ) : identifying tis through sequence alignment between orthologous genes has been described in ( 2 ) . briefly , it determines the tis of a gene by referring to its orthologous gene with known tis from other genera . it requires that they are aligned in the n - terminal region . in protisa , genes with tis labeled as ipt or cdc constitute the references to determine hsc tiss . we have been aware that errors in the ipt or cdc tiss might propagate into the hsc tiss via sequence alignment , especially among closely related genomes . to minimize such errors , a hsc tis is annotated only if it has support of orthologous genes from more than one different genus . med - start is a tis predictor with an iterative self - training algorithm based on a four - component statistical model to describe the tis in prokaryotic genomes ( 7 ) . the high performance of med - start has been demonstrated by evaluating on the e. coli and bacillus subtillis genomes . for the present work to computationally relocate tiss for large - scale genomes of both bacteria and archaea , several improvements used in our another work ( 9 ) have been made to the original algorithm of med - start . the modification first treated with the effects of the genomic background to recover regulatory motifs as well as to characterize the sequence patterns around the motifs and the start codon . further , the bias of the codon positional gc - content was applied to describe the coding potential of the context around a candidate start . details of the improved algorithm are available from http://ctb.pku.edu.cn/main/shegroup/medstartplus.htm the confirmed tiss via the above - mentioned evidences together with the improved predictions ( med ) then served as a refined annotation resource for the public database such as refseq . we implemented a meme - like algorithm to find signals upstream of tis ( 19 ) . it combines the positional weight matrix ( pwm ) of the signal , the distribution of the number of nucleotides between the signal and the tis ( spacer length ) , and the background nucleotide frequencies into a likelihood function . an em algorithm and a simulative annealing strategy were used to estimate the parameters . to classify the signals , we first included two kinds of typical signals as references , i.e. the widely accepted sd consensus we use the pwm of the 10 promoter in e. coli k-12 for the pribnow box ( 20 ) , while the pwm of the at - rich motif found in archaeoglobus fulgidus for the tata box . two scores were calculated for each signal , i.e. the sd score and the ta score . the former is calculated by matching the referenced consensus against the pwm of the signal , and the latter is measured by the euclidean distance between the pwm of the signal and the referenced pwm . interestingly , each score follows a bimodal distribution , which allows us to readily classify the signals into three categories : ( i ) ta - like , those resemble the pribnow ( or tata ) box ; ( ii ) sd - like , those resemble the sd signal and ( iii ) atypical , those resemble neither sd signal nor pribnow ( or tata ) box . we introduce a scoring function to measure the significance of a string as a signal comparing to a random background . a string is more likely to be a functional signal than a random sequence if the score > 0 . with the pwm and the spacer length distribution of the signal , we score each substring in the tis - upstream sequence and select the one with the highest score as the potential signal . confirmed tiss ( ipt , cdc and hsc ) were collected through database scanning , literature survey , conserved domain search and sequence alignment as follows : 
 imported ( ipt ) : a script was written to extract high - quality manual annotation in swiss - prot . init_met is used to indicate whether the initiator methionine has been cleaved off or not . finally , the ipt data set has been enriched by a simple n - terminal sequence comparison between closely related species.conserved domain confirmed ( cdc ) : the method to identify tis through conserved domain search is essentially similar to that in ( 6 ) . the tis for a gene with only one possible start codon upstream to the 5-most conserved domain hit was readily identified . to compensate for random matches , which would perhaps lead to an incorrect cdc - tis annotation , we removed six amino acids from the most upstream hit before processing ( 18 ) , since the frequency that a hit overlaps with the non - coding region by more than six amino acids is < 1% when examined on genes with ipt tiss.high similarity confirmed ( hsc ) : identifying tis through sequence alignment between orthologous genes has been described in ( 2 ) . briefly , it determines the tis of a gene by referring to its orthologous gene with known tis from other genera . it requires that they are aligned in the n - terminal region . in protisa , genes with tis labeled as ipt or cdc constitute the references to determine hsc tiss . we have been aware that errors in the ipt or cdc tiss might propagate into the hsc tiss via sequence alignment , especially among closely related genomes . to minimize such errors , a hsc tis is annotated only if it has support of orthologous genes from more than one different genus . imported ( ipt ) : a script was written to extract high - quality manual annotation in swiss - prot . init_met is used to indicate whether the initiator methionine has been cleaved off or not . finally , the ipt data set has been enriched by a simple n - terminal sequence comparison between closely related species . conserved domain confirmed ( cdc ) : the method to identify tis through conserved domain search is essentially similar to that in ( 6 ) . the tis for a gene with only one possible start codon upstream to the 5-most conserved domain hit was readily identified . to compensate for random matches , which would perhaps lead to an incorrect cdc - tis annotation , we removed six amino acids from the most upstream hit before processing ( 18 ) , since the frequency that a hit overlaps with the non - coding region by more than six amino acids is < 1% when examined on genes with ipt tiss . high similarity confirmed ( hsc ) : identifying tis through sequence alignment between orthologous genes has been described in ( 2 ) . briefly , it determines the tis of a gene by referring to its orthologous gene with known tis from other genera . it requires that they are aligned in the n - terminal region . in protisa , genes with tis labeled as ipt or cdc constitute the references to determine hsc tiss . we have been aware that errors in the ipt or cdc tiss might propagate into the hsc tiss via sequence alignment , especially among closely related genomes . to minimize such errors , a hsc tis is annotated only if it has support of orthologous genes from more than one different genus . med - start is a tis predictor with an iterative self - training algorithm based on a four - component statistical model to describe the tis in prokaryotic genomes ( 7 ) . the high performance of med - start has been demonstrated by evaluating on the e. coli and bacillus subtillis genomes . for the present work to computationally relocate tiss for large - scale genomes of both bacteria and archaea , several improvements used in our another work ( 9 ) have been made to the original algorithm of med - start . the modification first treated with the effects of the genomic background to recover regulatory motifs as well as to characterize the sequence patterns around the motifs and the start codon . further , the bias of the codon positional gc - content was applied to describe the coding potential of the context around a candidate start . details of the improved algorithm are available from http://ctb.pku.edu.cn/main/shegroup/medstartplus.htm the confirmed tiss via the above - mentioned evidences together with the improved predictions ( med ) then served as a refined annotation resource for the public database such as refseq . we implemented a meme - like algorithm to find signals upstream of tis ( 19 ) . it combines the positional weight matrix ( pwm ) of the signal , the distribution of the number of nucleotides between the signal and the tis ( spacer length ) , and the background nucleotide frequencies into a likelihood function . an em algorithm and a simulative annealing strategy were used to estimate the parameters . to classify the signals , we first included two kinds of typical signals as references , i.e. the widely accepted sd consensus we use the pwm of the 10 promoter in e. coli k-12 for the pribnow box ( 20 ) , while the pwm of the at - rich motif found in archaeoglobus fulgidus for the tata box . two scores were calculated for each signal , i.e. the sd score and the ta score . the former is calculated by matching the referenced consensus against the pwm of the signal , and the latter is measured by the euclidean distance between the pwm of the signal and the referenced pwm . interestingly , each score follows a bimodal distribution , which allows us to readily classify the signals into three categories : ( i ) ta - like , those resemble the pribnow ( or tata ) box ; ( ii ) sd - like , those resemble the sd signal and ( iii ) atypical , those resemble neither sd signal nor pribnow ( or tata ) box . a bayesian methodology is employed to predict potential signal upstream of each tis . we introduce a scoring function to measure the significance of a string as a signal comparing to a random background . a string is more likely to be a functional signal than a random sequence if the score > 0 . with the pwm and the spacer length distribution of the signal , we score each substring in the tis - upstream sequence and select the one with the highest score as the potential signal . as of july 2007 , protisa provides refined annotations for 440 genomes in refseq , with more than 390 000 confirmed tiss : ipt ( 8898 ) , cdc ( 302 192 ) and hsc ( 258 031 ) ( table 1 ) . the percentage of confirmed tiss in a genome varies from 9% to 73% with an average of 33% . the group gammaproteobacteria contributes to the majority of the data collection , which is much more evident for the ipt tiss . table 1.statistics of confirmed tiss ( as of july 2007)kingdomgroupgenome no.ipt no.cdc no.hsc no.gene no.archaeacrenarchaeota8207423814544729euryarchaeota2315613 175733015 389nanoarchaeota1015254163bacteriaacidobacteria2018369322213actinobacteria3628621 13712 56127 297aquificae13687350746bacteroidetes / chlorobi1110678254318516chlamydiae / verrucomicrobia112340522123858chloroflexi209117011099cyanobacteria2227713 4619 68216 855deinococcus - thermus499201112762603firmicutes8986463 64751 12277 320fusobacteria11773419837planctomycetes10439429655alphaproteobacteria5313831 92832 04345 656betaproteobacteria365223 89323 60134 828gammaproteobacteria1036 71693 83293 644127 165deltaproteobacteria14178932741611 996epsilonaproteobacteri1139605141436911spirochaetes923390625804635thermotogae18497292591other bacteria10499359663sum4408 898302 192258 031394 725number of genes with at least one confirmed tis . statistics of confirmed tiss ( as of july 2007 ) number of genes with at least one confirmed tis . a tis might be confirmed by several evidences . about 12% of the genes have more than one confirmed tis . transcriptional and translational signals are classified into three classes : sd - like , ta - like and atypical signals . of the 440 genomes , near half ( 212 ) were reported with only sd - like signals ( mainly in firmicutes and proteobacteria ) , 22 genomes with only atypical signals ( bacteroidetes / chlorobi and cyanobacteria ) . the other genomes were found with dual signals : sd - like and ta - like signals were found in 76 genomes ( actinobacteria and archaea ) , sd - like and atypical signals in 126 genomes ( proteobacteria ) and ta - like and atypical signals in four genomes ( table 2 ) . table 2.statistics of genomes with specific signals ( as of july 2007)kingdomgroupsd_like onlyatypical onlysd_like and ta_likesd_like and atypicalta_like and atypicalarchaeacrenarchaeota62euryarchaeota5162nanoarchaeota1bacteriaacidobacteria2actinobacteria1332aquificae1bacteroidetes / chlorobi65chlamydiae / verrucomicrobia110chloroflexi11cyanobacteria1111deinococcus - thermus4firmicutes7928fusobacteria1thermotogae1planctomycetes1alphaproteobacteria15137betaproteobacteria234gammaproteobacteria82120deltaproteobacteria923epsilonaproteobacteria11spirochaetes324otherbacteria1sum21222761264 statistics of genomes with specific signals ( as of july 2007 ) the basic functionalities aim to browse the stored data and to search the database with a user - specified input . the first section shows general information for a genome such as organism name , taxonomic group and genomic gc - content . this section also displays a sequence logo ( 21 ) and a histogram of the spacer length for each signal ( figure 1 ) . the second section contains tis annotation with start site and initiation signal for each gene . it shows the gene coordinate , gene identity ( pid and gene name ) , tis evidence type ( i.e. ipt or cdc or hsc or med ) , and the predicted signal . it also provides links to the evidence that supports the proposed tis to be confirmed : pmid or external database links for ipt tis , conserved domain search results for cdc - tis and multiple n - terminal sequence alignments among orthologous genes for hsc tis . figure 1.sequence logo and spacer length distribution of representative signals for the genomes ( a ) e. coli k-12 ; ( b ) s. coelicolor ; ( c ) a. fulgidus ; and ( c ) synechocystis sp . the positional weight matrix of the signal is visualized by a sequence logo in which the height of a letter on a given position is proportional to its occurring frequency . a letter is bottom - up shown if the occurring frequency is lower than that from the background . the spacer length is defined as the distance ( or the number of nucleotides ) between the tis and each of all annotated signals , which are calculated by the positional weight matrix visualized in sequence logo . sequence logo and spacer length distribution of representative signals for the genomes ( a ) e. coli k-12 ; ( b ) s. coelicolor ; ( c ) a. fulgidus ; and ( c ) synechocystis sp . the positional weight matrix of the signal is visualized by a sequence logo in which the height of a letter on a given position is proportional to its occurring frequency . a letter is bottom - up shown if the occurring frequency is lower than that from the background . the spacer length is defined as the distance ( or the number of nucleotides ) between the tis and each of all annotated signals , which are calculated by the positional weight matrix visualized in sequence logo . the webpage provides the user with a friendly interface to search the tis annotation by specifying a region in the genome sequence or by gene identifier such as name and pid . users can also specify the tis evidence types and compare the output with the refseq annotation . the annotation , based on which the web server is constructed , is available for download in batch . in addition , source codes ( written in c++ ) for the generation of cdc / hsc tis , the new version of med - start , and the motif finding algorithm are freely available in our website under the gnu gpl license . besides , referenced genes for hsc tis creation were compiled in a fasta file for download . despite the remarkable progresses made in computational annotation , there are continuous publications concerning tis annotation quality in the public database such as refseq ( 1,5,9,22,23 ) . a notable feature of protisa is the compilation of reliable tis by collecting evidences from experiments , literature , conserved domain search , sequence alignment and accurate prediction . it is interesting to apply the most reliable resource , ipt tiss , to estimate the reliability of cdc tiss , hsc tiss and med tiss . after removing redundancy from closely related genomes , we have collected a set of 3413 ipt tiss as benchmark , on which the cdc tiss report an accuracy of 99.4% and the hsc tiss report an accuracy of 99.0% . for med tiss predicted by the modified med - start algorithm it is argued that the signal upstream of tis usually implies the translation initiation mechanism ( 3,9,14,17,24 ) . another merit of protisa is the annotation of transcriptional and translational signals , which is visualized for each genome by a sequence logo for the signal content and a histogram for the spacer length distribution to tiss ( figure 1 ) . this would be helpful for biologists to speculate the initiation mechanism for a specific genome ( 14 ) . for example , in addition to the sd - like motif , we found in streptomyces coelicolor and tannnt motif that highly resembles the pribnow box reported previously , which generally locates at 10 bps upstream to the transcription start site ( tss ) ( 20 ) . moreover , the motif has a conserved position about 10 bps upstream to the tis , counting from tis to the 5 t in the  tannnt motif ( not shown in figure 1 ) . in other words , for some genes , the tss locates just a few base pairs upstream to or even overlaps with the tis , resulting in a leaderless gene . this would lead one to speculate the existence of initiation mechanism for leaderless gene in s. coelicolor , which is consistent with the results reported in ( 25 ) . interestingly , this motif was also found in several bacteria groups , for example actinobacteria , deinococcus - thermus and firmicutes , implying that leaderless gene may not be a marginal phenomenon as usually believed in terms of gene structure in bacteria ( 26 ) . an atypical signal is likely to be functional , especially given its conserved position to the tis . pcc 6803 a conserved dual - pyrimidine locating immediately upstream to the tiss ( figure 1d ) , which is consistent with the findings in ( 24 ) . such signal could serve as a target for biologists to decipher its regulation role by experiments , thus leading to a better understanding of the initiation mechanism . with the growing number of completely sequenced bacterial and archaeal genomes , the scientific value of a specific resource for tiss and the corresponding initiation signals is clear . we hope to increase the update frequency so that the database stays current as each new prokaryotic genome becomes available at ncbi . we plan to make protisa to be an evolving resource and add significant functionality over time . one direction of ongoing development is to explore the way of comparative genome analysis based on the divergent translation initiation mechanisms for bacteria and archaea . doing this is somewhat challenging since it is difficulty to develop a quantitative model to describe these complex mechanisms . protisa may also add items such as genes classification based on their initiation signals . to sum up , we believe that the resource will expand to suit the needs and requests of the research community for translation initiation studies .
correct annotation of translation initiation site ( tis ) is essential for both experiments and bioinformatics studies of prokaryotic translation initiation mechanism as well as understanding of gene regulation and gene structure . 
 here we describe a comprehensive database protisa , which collects tis confirmed through a variety of available evidences for prokaryotic genomes , including swiss - prot experiments record , literature , conserved domain hits and sequence alignment between orthologous genes . 
 moreover , by combining the predictions from our recently developed tis post - processor , protisa provides a refined annotation for the public database refseq . 
 furthermore , the database annotates the potential regulatory signals associated with translation initiation at the tis upstream region . as of july 2007 
 , protisa includes 440 microbial genomes with more than 390 000 confirmed tiss . 
 the database is available at http://mech.ctb.pku.edu.cn/protisa