After a long week, the Fredrick lab enjoyed a nice happy hour at Budd Dairy Food Hall in downtown Columbus.
Author: warner.474
New publication in Nucleic Acids Research! April 2023
Abstract
Ribosome biogenesis occurs co-transcriptionally and entails rRNA folding, r protein binding, rRNA processing, and rRNA modification. In most bacteria, the 16S, 23S, and 5S rRNAs are co-transcribed, often with one or more tRNAs. Transcription involves a modified RNA polymerase, called the antitermination complex, which forms in response to cis-acting elements (boxB, boxA, and boxC) in the nascent pre-rRNA. Sequences flanking the rRNAs are complementary and form long helices known as leader-trailer helices. Here, we employed an orthogonal translation system to interrogate the functional roles of these RNA elements in 30S subunit biogenesis in Escherichia coli. Mutations that disrupt the leader-trailer helix caused complete loss of translation activity, indicating that this helix is absolutely essential for active subunit formation in the cell. Mutations of boxA also reduced translation activity, but by only 2- to 3-fold, suggesting a smaller role for the antitermination complex. Similarly modest drops in activity were seen upon deletion of either or both of two leader helices, termed here hA and hB. Interestingly, subunits formed in the absence of these leader features exhibited defects in translational fidelity. These data suggest that the antitermination complex and precursor RNA elements help to ensure quality control during ribosome biogenesis.
Paper published in Nucleic Acids Research! February 2023
Ribosome lacking bS21 gain function to regulate protein synthesis in Flavobacterium johnsoniae.
Zakkary A. McNutt, Bappaditya Roy, Bryan T. Gemler, Elan A. Shatoff, Kyung-Mee Moon, Leonard J. Foster, Ralf Bundschuh and Kurt Fredrick
ABSTRACT
Ribosomes of Bacteroidia (formerly Bacteroidetes) fail to recognize Shine-Dalgarno (SD) sequences even though they harbor the anti-SD (ASD) of 16S rRNA. Inhibition of SD-ASD pairing is due to sequestration of the 3’ tail of 16S rRNA in a pocket formed by bS21, bS18, and bS6 on the 30S platform. Interestingly, in many Flavobacteriales, the gene encoding bS21, rpsU, contains an extended SD sequence. In this work, we present genetic and biochemical evidence that bS21 synthesis in Flavobacterium johnsoniae is autoregulated via a subpopulation of ribosomes that specifically lack bS21. Mutation or depletion of bS21 in the cell increases translation of reporters with strong SD sequences, such as rpsU’-gfp, but has no effect on other reporters. Purified ribosomes lacking bS21 (or its C-terminal region) exhibit higher rates of initiation on rpsUmRNA and lower rates of initiation on other (SD-less) mRNAs than control ribosomes. The mechanism of autoregulation depends on extensive pairing between mRNA and 16S rRNA, and exceptionally strong SD sequences, with predicted pairing free energies of < –13 kcal/mol, are characteristic of rpsU across the Bacteroidota. This work uncovers a clear example of specialized ribosomes in bacteria.
Ribosome: Structure and Function Meeting in Bordeaux, France
The Fredrick Lab made the trip out to Bordeaux, France to attend the Ribosome meeting (@2022Ribosomes). Kurt gave a talk, while Sepideh, Siddik, and Ben gave poster presentations. We were joined by our collaborator and fellow buckeye, Ralf Bundschuh. Everyone got great feedback and talked a lot of science! Not to mention there was a lot of good wine and food there too!
