Abstract
The anti-Shine-Dalgarno (ASD) sequence of 16S rRNA is highly conserved across Bacteria, and yet usage of Shine-Dalgarno (SD) sequences in mRNA varies dramatically, depending on the lineage. Here, we compared the effects of ASD mutagenesis in Escherichia coli, a Gammaproteobacteria which commonly employs SD sequences, and Flavobacterium johnsoniae, a Bacteroidia which rarely does. In E. coli, 30S subunits carrying any single substitution at positions 1,535–1,539 confer dominant negative phenotypes, whereas subunits with mutations at positions 1,540–1,542 are sufficient to support cell growth. These data suggest that CCUCC (1,535–1,539) represents the functional core of the element in E. coli. In F. johnsoniae, deletion of three ribosomal RNA (rrn) operons slowed growth substantially, a phenotype largely rescued by a plasmid-borne copy of the rrn operon. Using this complementation system, we found that subunits with single mutations at positions 1,535–1,537 are as active as control subunits, in sharp contrast to the E. coli results. Moreover, subunits with quadruple substitution or complete replacement of the ASD retain substantial, albeit reduced, activity. Sedimentation analysis revealed that these mutant subunits are overrepresented in the subunit fractions and underrepresented in polysome fractions, suggesting some defect in 30S biogenesis and/or translation initiation. Nonetheless, our collective data indicate that the ASD plays a much smaller role in F. johnsoniae than in E. coli, consistent with SD usage in the two organisms.
Highlights
Faithful protein synthesis requires that the translation machinery select the correct start codon over other AUG or similar trinucleotides
The ASD region of the E. coli ribosome has been targeted in several previous studies (Hui and de Boer, 1987; Jacob et al, 1987; Lee et al, 1996; Rackham and Chin, 2005; Hui et al, 1988; Yassin et al, 2005)
Certain variants (C1536G, U1537G, C1538G, C1539A, and C1539U) were especially deleterious. These strong effects stem from altered specificity of the mutant ribosomes during initiation, consistent with widespread proteomic changes seen in analogous studies (Jacob et al, 1987)
Summary
Faithful protein synthesis requires that the translation machinery select the correct start codon over other AUG or similar trinucleotides. Intrinsic sequence and structural features of the mRNA enable start codon recognition. One well-known feature in prokaryotic cells is the ShineDalgarno (SD) sequence, a purine-rich element that lies upstream from the start codon and can pair with the anti-SD (ASD) sequence contained in the 3’ tail of 16S rRNA (Shine and Dalgarno, 1974; Steitz and Jakes, 1975). There are many mRNAs that naturally lack a SD and yet are accurately and efficiently translated, indicating that other features of mRNA can direct start codon selection (Espah Borujeni et al, 2014; Li et al, 2014; Hockenberry et al, 2017)
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