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Materials and methods

Genome sequence data

Genome sequences and their annotations were obtained from GenBank [Benson et al. 2002].

Genomes	Accession No.
A. fulgidus	AE000782
B. burgdorferi	AE000783
B. halodurans	BA000004
B. subtilis	AL009126
Buchnera sp.	AP000398
C. acetobutylicum	AE001437
C. jejuni	AL111168
C. pneumoniae	AE001363
D. radiodurans chromosome 1	AE000513
E. coli K-12	U00096
H. influenzae	L42023
H. pylori J99	AE001439
L. innocua	AL592022
L. lactis	AE005176
M. genitalium	L43967
M. jannaschii	L77117
M. tuberculosis	AL123456
N. meningitidis MC58	AE002098
P. abyssi	AL096836
P. aeruginosa	AE004091
S. aureus Mu50	BA000017
S. pneumoniae	AE005672
Synechocystis sp.	AB001339
T. maritima	AE000512
V. cholerae chromosome I	AE003852
X. fastidiosa	AE003849

RNA folding and filters

For each gene in a genome, we collected upstream sequence segments up to 300 residues or the neighboring ORF. ORFs with less than 50 amino acids were considered as intergenic regions since some attenuation mechanisms are coupled with the synthesis of leader peptides. The total number of these segments was 3560 in B. subtilis and 3613 in E. coli. Stem-loop structures in these segments were predicted by using the RNAfold program [Hofacker et al. 1994]. As a reference, each upstream sequence was shuffled to produce random sequences with the same base composition and folded in the same manner. Using the characteristics of the known structures, we derived three filters based on location and poly-U runs which were applied to the collected folds to optimize the possibility of finding new attenuation structures. The filtering process retained 43 of the 46 known attenuation terminators. The three filters used, based on known attenuation terminators in B. subtilis and E. coli are: (i) Poly-U stretches (>=4 Us) must be located within from the 10 residues at the top of the stem to 15 residues downstream of the stem: (ii) the length of the 3' sequence from the end of the stem to the start position of the downstream gene must be <=170 and (iii) the length of the 5' sequence from the beginning of the intergenic region to the 5' start of the stem must be >=30, if the upstream gene is in the same orientation as the downstream (to partially eliminate 'standard' transcription terminator structures).

Figure. Definition of filters.
Promoter prediction

To further support the prediction of attenuation terminator, we also appliced promoter prediction: if a promoter is predicted in the upstream of a predicted attenuation terminator, then it is more likely to be the real attenuation terminator. NNPP version 2.2 [Reese MG 2001, http://www.fruitfly.org/seq_tools/promoter.html] was used for the prediction of prokaryotic promoters.
Significance measurement

To evaluate the significance of stem-loop structures in upstream sequences, we used the distribution of those structures in the randomly shuffled sequences, which has the same base composition and only the order of the bases are randomized. First, all the stem-loop structures found in the shuffled sequences are plotted according to their stability and stem length. Then the line running along the largest variance is calculated by principal component analysis [Afifi and Clark 1996]. Using the standard deviation which is calculated from distribution of stem-loop structures in the shuffled sequence around the line, Z-score is calculated for each stem-loop structure. We took those stem-loop structures in the upstream sequences with Z<=-2 as significant structures.
Identification of orthologous genes

To identify orthologous gene pair among a pair of genomes, first we carried out all-against-all comparison between the sets of proteins, each of which is from a whole genome. We used the BLASTP program [Altschul et al. 1997] for this comparison. Only the hits with BLAST E<=0.001 are collected as significant hits. Then, among those significant hits, a pair of genes are defined as ortholog if the pair satisfies the "bi-directional best hit" [Snel et al. 2000]. For a group of more than 2 genomes, a group of genes, each of which is from a genome, are defined as ortholog if all possible pairs of genes satisfy the bi-directional significant best hit.
References
- Afifi AA, Clark V: Computer-aided multivariate analysis. 3rd ed. 1996, Chapman & Hall, Baca Raton, Florida.
- Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997, 25:3389-3402.
- Benson DA, Karsch-Mizrachi I, Lipman DJ, Ostell J, Rapp BA, Wheeler DL: GenBank. Nucleic Acids Res 2002, 30:17-20.
- Hofacker IL, Fontana W, Stadler PF, Bonhoeffer S, Tacker M, Schuster P: Fast folding and comparison of RNA secondary structures. Monatsh Chem 1994, 125:167-188.
- Reese MG: Application of a time-delay neural network to promoter annotation in the Drosophila melanogaster genome. Comput Chem 2001, 26:51-56.
- Snel B, Lehmann G, Bork P, Huynen MA: STRING: a web-server to retrieve and display repeatedly occurring neighbourhood of a gene. Nucleic Acids Res 2000, 28:3442-3444.