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Access Type

WSU Access

Date of Award

January 2021

Degree Type


Degree Name



Biological Sciences

First Advisor

Jared Schrader


Bacterial translation comprising of three steps: initiation, elongation and termination is a highly energy intensive step, of which translation initiation is very important for fidelity of gene expression. This is because the canonical start codon, AUG, complements both initiator and elongator methionyl-tRNAs making it important for ribosome to distinguish the start AUG codon from elongator AUG codons which is thought to be mediated by base pairing of the Shine-Dalgarno (SD) sequence in the mRNA’s 5′ UTR and the complementary anti-SD sequence in the 16S rRNA of the 30S subunit of ribosome. However, this mechanism was proposed based on the sequence information of few E.coli phage mRNAs and 16S rRNA sequence of E.coli. The complementary anti-SD sequence was found to be present in E.coli and few other bacteria of which 16S rRNA was later sequenced, and therefore, this mechanism was assumed to be present universally. However, with advancement in sequencing technologies, several high-throughput studies have showed that SD:Anti-SD base pairing is not essential for start codon selection and transcriptomics has revealed that non-SD mRNAs (which lack a SD sequence in the 5′ UTR) and leaderless mRNAs (which completely lack any 5′ UTR), are broadly distributed across bacteria including the model organism Caulobacter crescentus (which contains only 30% of mRNAs having SD) and can initiate translation in the absence of the SD sequence. Hence, to explain how the mRNA translation initiates in the absence of SD sequence, another mechanism “unique accessibility hypothesis” which assumes that the TIRs are more accessible (less RNA secondary structure) as compared to the other regions of the mRNA has been proposed and tested (computationally and experimentally) in various organisms. To test this hypothesis in C. crescentus, we performed computational analysis to compute the accessibility of the TIRs and other regions of mRNAs using a metric (ΔGunfold) which revealed similar observations indicating importance of TIR accessibility for translation initiation and correct start codon selection. Further, to systematically test the effects of accessibility and other factors on leaderless (start codon identity and presence of short leaders) and leadered mRNA translation initiation (presence of SD sequences) which is not looked into, synthetic in vivo translation initiation reporters were designed that ensured translation of the reporter gene only from the AUG codon in the TIR allowing the control of the TIR region. Translation efficiency values calculated using RNA-seq and ribosome profiling data were used to test the effect of these factors on translation initiation of natural mRNAs. Using these data, it was found that start codon accessibility is a key determinant in correct start codon selection and initiating translation from the TIR, while leader length and start codon identity impacts leaderless mRNA translation initiation efficiency, and presence of SD influences leadered mRNA translation initiation efficiency in bacteria.

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