Prediction of transcriptional terminators in Bacillus subtilis and related species

Abstract

In prokaryotes, genes belonging to the same operon are transcribed in a single mRNA molecule. Transcription starts as the RNA polymerase binds to the promoter and continues until it reaches a transcriptional terminator. Some terminators rely on the presence of the Rho protein, whereas others function independently of Rho. Such Rho-independent terminators consist of an inverted repeat followed by a stretch of thymine residues, allowing us to predict their presence directly from the DNA sequence. Unlike in Escherichia coli, the Rho protein is dispensable in Bacillus subtilis, suggesting a limited role for Rho-dependent termination in this organism and possibly in other Firmicutes. We analyzed 463 experimentally known terminating sequences in B. subtilis and found a decision rule to distinguish Rho-independent transcriptional terminators from non-terminating sequences. The decision rule allowed us to find the boundaries of operons in B. subtilis with a sensitivity and specificity of about 94%. Using the same decision rule, we found an average sensitivity of 94% for 57 bacteria belonging to the Firmicutes phylum, and a considerably lower sensitivity for other bacteria. Our analysis shows that Rho-independent termination is dominant for Firmicutes in general, and that the properties of the transcriptional terminators are conserved. Terminator prediction can be used to reliably predict the operon structure in these organisms, even in the absence of experimentally known operons. Genome-wide predictions of Rho-independent terminators for the 57 Firmicutes are available in the Supporting Information section.

Figure 1. Distribution of the Gibbs Free Energy of Stem-Loop Formation

The distribution is calculated from 425 experimentally identified transcriptional terminators in B. subtilis. The dotted curve shows the distribution for E. coli, as calculated from 147 previously collected Rho-independent terminator sequences in this organism [16].

Figure 2. Distribution of the Length of the Stem in Nucleotides

The distribution is calculated from 425 transcriptional terminators in B. subtilis and 147 previously published Rho-independent terminators in E. coli [16].

Figure 3. Distribution of Gibbs Free Energy of Stem-Loop Formation, Divided by the Length of the Stem-Loop Structure in Nucleotides

The distribution is calculated from 425 transcriptional terminators in B. subtilis, and 147 previously published Rho-independent terminators in E. coli [16].

Figure 4. Distribution of the Number of Thymine Residues in the 15 Base Pair T-Stretch following the Stem Loop

The distribution is calculated from 425 transcriptional terminators in B. subtilis, and 147 previously published Rho-independent terminators in E. coli [16].

Figure 5. Distribution of the Number of Residues in the Loop of Rho-Independent Terminators

The distribution is calculated from 425 transcriptional terminators in B. subtilis and 147 previously collected Rho-independent terminator sequences in E. coli [16].

Figure 6. Distribution of the Position of B. subtilis Rho-Independent Terminators with Respect to the Stop Codon of the Last Gene in the Operon

The distance between the first nucleotide of the stem-loop and the last nucleotide of the stop codon is shown.

Figure 7. The yqfSU Operon in B. subtilis Consists of the Two Genes yqfS and yqfU, Separated by the Intervening Gene yqfT, Located on the Opposite Strand

The terminator sequence downstream of yqfT is virtually identical to the complementary sequence on the opposite strand. However, a Northern blotting experiment [27] revealed that the complementary sequence does not act as a transcriptional terminator. Arrows indicate transcription start sites; stem-loops represent transcriptional terminators.

Figure 8. Sensitivity of Predicting Transcriptional Terminators, Evaluated for 57 Firmicutes and 29 Other Bacterial Species

Firmicutes are shown in dark gray; other bacterial species are shown in light gray. (+), (−), or (0) in front of the organism name denotes that the organism is Gram-positive, Gram-negative, or lacks a cell wall, respectively.

Figure 9. Average Gibbs Free Energy of Stem-Loop Formation and the Average Number of Thymine Residues in the T-Stretch

These are calculated from the predicted Rho-independent terminators in the 82 bacterial species we consider. Circles represent organisms belonging to the Firmicutes phylum; crosses represent other bacterial species.