Replication-transcription collisions impose DNA strand-specific constraints on gene length in bacteria

Gene length is a peculiar genomic feature that exhibits minimal variation within domains of life, suggesting universal underlying constraints. The length of a gene is primarily influenced by its function, expression level, and mutation risk. Interestingly, many of these factors vary depending on whether the gene is located on the leading or lagging strand of DNA replication. Studies in bacterial species E. coli and B. subtilis have shown that genes tend to be shorter on the lagging strand, potentially reflecting selection to minimize head-on encounters between the replication and transcription machinery. However, the universality of strand-specific constraints on gene length and the evolutionary basis remain unexplored. Here, using comparative genomics, we analyzed gene lengths in the bacterial domain and revealed a non-neutral distribution of gene lengths across leading and lagging strands. Genes and operons are consistently shorter on the lagging strand, irrespective of essentiality or functionality. The length restriction was more pronounced in bacterial species with a dual DNA polymerase mode of replication, which may experience severe head-on collisions between replication and transcription. Remarkably, we found that with increasing length of transcription units, substitution rates increased in the promoters on the lagging strand rather than the coding sequences, revealing a length-dependent and lagging strand-specific cis-regulatory mutational susceptibility. Together, we uncovered a pervasive selection pressure that optimizes gene lengths in a DNA strand-specific manner across bacteria to preserve the genetic integrity of promoters.