Deciphering the influence of genome architecture in minimized bacteria

Stochastic gene expression begins with the repeated transcription of DNA by RNA polymerase (RNAP) to create the various functional forms (rRNA, tRNA, mRNA, and sRNA) of the bacterial transcriptome. The initiation and termination of transcriptional events are associated with the genome architecture, the local arrangement of genetic features along the circular bacterial genome identified via sequence motifs (promoters, Shine-Dalgarno, gene open-reading frames, and transcription termination sites, etc.). The local arrangement of these sequence motifs form operon-like regions, known as transcriptional units, directly controlling the expression of encoded RNA species. The stochastic activity between RNAP and the genetic features of a transcription unit results in differential transcription forming RNA isoforms, various RNA transcripts from a similar region of different lengths. Locations of the genetic features that define the transcription unit boundaries were predicted using structural and bioinformatic analysis, and then integrated to predict transcriptional units within two minimized bacteria, JCVI-syn1.0 and JCVI-syn3A. The theoretical predictions agree well with observations of expression obtained using Oxford Nanopore Technologies Direct RNA sequencing for Syn1.0 the precursor of Syn3A. Comparisons of the computational and experimental results help to provide new insight into transcriptional activity within the organisms, resolve the distribution of RNA isoform complexity evolved from key genomic regions such as those involved in membrane transport and cell division, motivate the investigation into the post-transcriptional activity of RNA, and explain the impact on gene expression from the genome reduction of Syn1.0 to Syn3A.