No abstract

Caulobacter crescentus is a model alphaproteobacterium with a well-studied genetic network controlling its cell cycle. Essential for such studies is an accurate map of the expressed features of its genome. Here, we provide an updated map of the expressed RNAs by integrative analysis of 5′ global rapid amplification of cDNA ends, transcriptome sequencing, rifampicin treatment RNA sequencing, and RNA end-enriched sequencing data sets.

Bacterial translation is thought to initiate by base pairing of the 16S rRNA and the Shine–Dalgarno sequence in the mRNA’s 5′ untranslated region (UTR). However, transcriptomics has revealed that leaderless mRNAs, which completely lack any 5′ UTR, are broadly distributed across bacteria and can initiate translation in the absence of the Shine–Dalgarno sequence. To investigate the mechanism of leaderless mRNA translation initiation, synthetic in vivo translation reporters were designed that systematically tested the effects of start codon accessibility, leader length, and start codon identity on leaderless mRNA translation initiation. Using these data, a simple computational model was built based on the combinatorial relationship of these mRNA features that can accurately classify leaderless mRNAs and predict the translation initiation efficiency of leaderless mRNAs. Thus, start codon accessibility, leader length, and start codon identity combine to define leaderless mRNA translation initiation in bacteria.

Translation initiation is an essential step for fidelity of gene expression, in which the ribosome must bind to the translation initiation region (TIR) and position the initiator tRNA in the P-site (1). For this to occur correctly, the TIR encompassing the ribosome binding site (RBS) needs to be highly accessible (2-5). ΔGunfold is a metric for computing accessibility of the TIR, but there is no automated way to compute it manually with existing software/tools limiting throughput. ΔGunfold leaderless allows users to automate the ΔGunfold calculation to perform high-throughput analysis. Importantly, ΔGunfold leaderless allows calculation of TIRs of both leadered mRNAs and leaderless mRNAs which lack a 5' UTR and which are abundant in bacterial, archaeal, and mitochondrial transcriptomes (4, 6, 7). The ability to analyze leaderless mRNAs also allows one additional feature where users can computationally optimize leaderless mRNA TIRs to maximize their gene expression (8, 9). The ΔGunfold leaderless package facilitates high-throughput calculations of TIR accessibility, is designed to calculate TIR accessibility for leadered and leaderless mRNA TIRs which are abundant in bacterial/archaeal/organellar transcriptomes and allows optimization of leaderless mRNA TIRs for biotechnology.

Bacterial translation is thought to initiate by base-pairing of the 16S rRNA and the Shine-Dalgarno sequence in the mRNA's 5′ UTR. However, transcriptomics has revealed that leaderless mRNAs, which completely lack any 5′ UTR, are broadly distributed across bacteria and can initiate translation in the absence of the Shine-Dalgarno sequence. To investigate the mechanism of leaderless mRNA translation initiation, synthetic in vivo translation reporters were designed that systematically tested the effects of start codon accessibility, leader length, and start codon identity on leaderless mRNA translation initiation. Using this data, a simple computational model was built based on the combinatorial relationship of these mRNA features which can accurately classify leaderless mRNAs and predict the translation initiation efficiency of leaderless mRNAs. Thus, start codon accessibility, leader length, and start codon identity combine to define leaderless mRNA translation initiation in bacteria.Translation initiation is a critical step for fidelity of gene expression in which the ribosome initiation complex is formed on the start codon of the mRNA. Since the canonical start codon, AUG, compliments both initiator and elongator methionyl-tRNAs, the ribosome must distinguish the start AUG codon from elongator AUG codons. Incorrect initiation at an elongator AUG can lead to non-functional products that can be detrimental to cellular fitness (1-3).Canonical start codon selection is thought to occur by the base-pairing of the 16S rRNA with a Shine-Dalgarno (SD) sequence in the mRNA located 5nt upstream of the start codon (4-6). The base pairing between the 16S rRNA and mRNA was shown to be critical for initiation since mutation of the anti-SD (aSD) in the 16S rRNA is lethal (7), and translation of a gene lacking a canonical SD sequence could be restored when the 16S of the rRNA were mutated to a complimentary sequence (8). While the SD-aSD pairing clearly impacts translation initiation efficiency (TIE) in E. coli, other studies have found that the SD:aSD interaction is not essential for correct selection of the start codon (9,10). Indeed, "orthogonal" ribosomes with altered 16S rRNA aSD sequences were found to initiate at the normal start codons throughout the transcriptome (11). Interestingly, E. coli lacks SD sites within its genome in approximately 30% of its translation initiation regions (TIRs) with other species of bacteria containing SD sites in as few as 8% of their TIRs (12,13). Indeed, RNA-seq based transcription mapping experiments have found that many bacterial mRNAs are "leaderless" and begin directly at the AUG start codon (14-16), and that these mRNAs are abundant in pathogens such as M. tuberculosis and in the mammalian mitochondria (17)..To account for the lack of essentiality of the SD site, a "Unique accessibility model" was proposed which posited that start codon selection occurs due to the TIR being accessible to 3 initiating ribosomes, while elongator AUGs are physically inaccessible due to RNA secondary str...