Poster abstracts
Poster number 14 submitted by Mohammed Husain Bharmal
Probing determinants of translation initiation in non-SD mRNAs
Mohammed Husain Bharmal (Biological Sciences, Wayne State university), Jared Schrader (Biological Sciences, Wayne State university)
Abstract:
A key step in gene expression is when ribosomes initiate mRNA at the start codon. In E. coli, translation initiation at the start codon is facilitated by base pairing of the 16S rRNA and a Shine-Dalgarno (SD) site in the mRNA. Surprisingly, recent genome surveys revealed that only half of bacterial genes contain SD sequences, with some bacterial species having as few as 8% of their genes encoded with upstream SD sequences1. To understand the mechanism(s) of non-SD translation initiation, we utilize Caulobacter crescentus, an α-proteobacterium that is highly adapted to initiation in leadered non-SD mRNAs (lacking SD sequence in 5’ untranslated region (UTR)) and leaderless mRNAs (lacking 5’ UTRs)2. We hypothesize that a lack in mRNA secondary structure increases the accessibility of the start AUG codon to the ribosome thereby facilitating initiation. To test this hypothesis, we used computational analysis to predict start codon regional accessibility by calculating ΔGunfold across the Caulobacter genome. The ΔGunfold predictions revealed that the start AUG codons are more accessible than elongating AUG codons within the body of mRNAs. Interestingly, leaderless mRNAs have a lower ΔGunfold than leadered RNAs, and generation of a set of synthetic leaderless RNAs with larger ΔGunfold strongly inhibits leaderless translation. Mutations in the 5′ UTR’s of SD and non-SD mRNAs revealed that ΔGunfold negatively correlates with translation levels although to a lower extent. To further establish the role of mRNA structure and start codon selection we are generating large ribosome binding site (RBS) libraries across diverse mRNAs with altered secondary structures whose translation will be assayed by flow-seq3.
References:
References:
1. Chang, B.; Halgamuge, S.; Tang, S. L., Analysis of SD sequences in completed microbial genomes: Non-SD-led genes are as common as SD-led genes. Gene 2006, 373, 90-99.
2. Schrader, J. M.; Zhou, B.; Li, G. W.; Lasker, K.; Childers, W. S.; Williams, B.; Long, T.; Crosson, S.; McAdams, H. H.; Weissman, J. S.; Shapiro, L., The coding and noncoding architecture of the Caulobacter crescentus genome. PLoS Genet 2014, 10 (7), e1004463.
3. Kosuri, S.; Goodman, D. B.; Cambray, G.; Mutalik, V. K.; Gao, Y.; Arkin, A. P.; Endy, D.; Church, G. M., Composability of regulatory sequences controlling transcription and translation in Escherichia coli. Proc. Natl. Acad. Sci. U. S. A. 2013, 110 (34), 14024-9.
Keywords: non-SD translation initiation, start codon accessibility