Kurt Fredrick

Leader-trailer helices of precursor rRNAs are ubiquitous in bacteria and archaea and are crucial for ribosome biogenesis in Escherichia coli. Here, using an orthogonal ribosome system, we investigate whether an engineered protein-aptamer bridge can functionally substitute for the leader-trailer helix (hLT) of pre-16S rRNA in E. coli. We find that components of such a bridge, and even the 3’ aptamer array component alone, can increase 30S subunit formation in the absence of hLT. Loss of RNase R or mutations of the degradosome (RNase E, PNPase, or RhlB) also boost 30S biogenesis in the absence of hLT. Quantification of orthogonal 16S rRNA suggests that these alterations act by stabilizing pre-16S rRNA molecules lacking the leader or trailer, giving sufficient time for rRNA folding to occur. In all cases, 16S rRNA level correlates with 30S subunit activity; however, the active fraction of 30S particles formed is substantially higher in the presence of hLT than in its absence. Notably, the complete set of aptamer-bridge components can fully stabilize pre-16S rRNA lacking hLT but fails to increase the active fraction of 30S particles formed. These findings suggest that hLT enhances both the speed and efficiency of 30S subunit formation in the cell.

Bacteroidia ribosomes are “blind” to Shine–Dalgarno (SD) sequences because the anti-SD (ASD) of 16S rRNA is sequestered by bS21, bS18, and bS6 on the 30S platform. In Flavobacterium johnsoniae, one gene contains a strong SD sequence—rpsU, which encodes bS21. Flavobacterium johnsoniae ribosomes lacking bS21 translate rpsU at a higher rate, which establishes an autoregulatory cycle. Here, we targeted the ASD of 16S rRNA in F. johnsoniae, ablating the core element (CCUCC to GAAGC). Replacement of each 16S gene with this quadruple-substituted (QS) allele had little effect until the last gene was changed. The final strain, containing only QS ribosomes, grows poorly. This defect can be largely rescued by replacing the translation initiation region (TIR) of rpsU with the SD-less TIR of tuf. Purified QS ribosomes fail to translate native rpsU messenger RNA (mRNA) but are active on SD-less mRNAs. We also selected suppressors of the ASD-ablated strain, many of which carried a point mutation in the SD of rpsU. Such mutations increase bS21 synthesis in the ASD-ablated strain but reduce bS21 synthesis by wild-type ribosomes, underscoring the importance of SD–ASD pairing in the natural case. We also find that ASD ablation inhibits rRNA processing, an effect independent of bS21.

Bacteria of class Bacteroidia lack Shine-Dalgarno (SD) sequences and instead rely on other messenger RNA (mRNA) features, including upstream adenines, for start codon selection. Bacteroidia ribosomes contain the anti-SD (ASD) sequence of 16S ribosomal RNA (rRNA) but are “blind” to SD sequences. This occurs due to the sequestration of the ASD through interactions with bS21, bS18, and bS6 on the 30S platform domain. In many Bacteroidia, including Flavobacterium johnsoniae, there is one gene with an extended SD—rpsU, which encodes bS21. Ribosomes lacking bS21 exhibit high-level translation of rpsU, establishing an autoregulatory circuit in the cell. In this work, we investigate the structural basis of initiation on rpsU mRNA. We find using cryo-electron microscopy that initiation entails the formation of a 13-base pair SD–ASD helix that sterically occludes bS21. Mutations of bS21, bS18, or bS6 that compromise the platform pocket liberate the 3′ tail of 16S rRNA, enable SD–ASD pairing, and enhance initiation. As initiation on rpsU mRNA depends on SD–ASD pairing, we infer that dissociation of bS21 from replete ribosomes limits their initiation rate. This work shows how a compositional change of the ribosome can govern translation of a specific gene.

Previous studies of the 70S ribosome from Flavobacterium johnsoniae revealed a novel ribosomal protein, bL38, which interacts with uL6 on the 50S subunit. This 5.6 kDa protein is conserved across the Bacteroidia and encoded downstream of bL28 and bL33 in a three-gene operon. Here, we show that bL38 is critical for the growth of F. johnsoniae, and depletion of bL38 leads to accumulation of immature 50S particles, which lack uL6 and retain precursor rRNA sequences. Cryo-EM analysis of these particles reveals several putative assembly intermediates, all showing an absence of electron density for uL6 and the entire uL12 stalk region and additional densities corresponding to the unprocessed ends of the pre-23S rRNA. Extra copies of the uL6 gene can rescue the phenotypes caused by bL38 depletion, suggesting that bL38 facilitates uL6 incorporation during 50S subunit biogenesis. Cryo-EM analysis of 50S particles from this rescued strain reveals nearly twice as many intermediates, suggesting a broader and more robust assembly landscape. Differential scanning fluorimetry shows that uL6 of F. johnsoniae is intrinsically unstable, and bL38 increases the melting temperature of uL6 by 12°C. Collectively, these data suggest that bL38 binds and stabilizes uL6, thereby promoting 50S biogenesis in the Bacteroidia.

The sarcin-ricin loop (SRL) is one of the most conserved segments of ribosomal RNA (rRNA). Translational GTPases (trGTPases), such as EF-G, EF-Tu, and IF2, form contacts with the SRL that are critical for GTP hydrolysis and factor function. Previous studies showed that expression of 23S rRNA lacking the SRL confers a dominant lethal phenotype inEscherichia coli. Isolated ΔSRL particles were found to be not only inactive in protein synthesis but also incompletely assembled. In particular, block 4 of the subunit, which includes the peptidyl transferase center, remained unfolded. Here, we explore the basis of this assembly defect. We find that 23S rRNA extracted from ΔSRL subunits can be efficiently reconstituted into 50S subunits, and these reconstituted ΔSRL particles exhibit full peptidyl transferase activity. We also further characterize ΔSRL particles purified from cells, using cryo-EM and proteomic methods. These particles lack density for rRNA and r-proteins of block 4, consistent with earlier chemical probing data. Incubation of these particles with excess total r-protein of the large subunit (TP50) fails to restore substantial peptidyl transferase activity. Interestingly, proteomic analysis of control and mutant particles shows an overrepresentation of multiple assembly factors in the ΔSRL case. We propose that one or more GTPases normally act to release assembly factors, and this activity is blocked in the absence of the SRL.

Ribosomal RNAs are transcribed as part of larger precursor molecules. InEscherichia coli, complementary RNA segments flank each rRNA and form long leader–trailer (LT) helices, which are crucial for subunit biogenesis in the cell. A previous study of 15 representative species suggested that most but not all prokaryotes contain LT helices. Here, we use a combination of in silico folding and covariation methods to identify and characterize LT helices in 4464 bacterial and 260 archaeal organisms. Our results suggest that LT helices are present in all phyla, including Deinococcota, which had previously been suspected to lack LT helices. In very few organisms, our pipeline failed to detect LT helices for both 16S and 23S rRNA. However, a closer case-by-case look revealed that LT helices are indeed present but escaped initial detection. Over 3600 secondary structure models, many well supported by nucleotide covariation, were generated. These structures show a high degree of diversity. Yet, all exhibit extensive base-pairing between the leader and trailer strands, in line with a common and essential function.

30S subunits become inactive upon exposure to low Mg2+ concentration, due to a reversible conformational change that entails nucleotides (nt) in the neck helix (h28) and 3’ tail of 16S rRNA. This active-to-inactive transition involves partial unwinding of h28 and re-pairing of nt 921-923 with nt 1532-1534, which requires flipping of the 3’ tail by ~180 degrees. Growing evidence suggests that immature 30S particles adopt the inactive conformation in the cell, and transition to the active state occurs at a late stage of maturation. Here, we target nucleotides that form the alternative helix (hALT) of the inactive state. Using an orthogonal ribosome system, we find that disruption of hALT decreases translation activity in the cell modestly, by ~2-fold, without compromising ribosome fidelity. Ribosomes carrying substitutions at positions 1532-1533 support growth of E. coli strain Δ7 prrn (which carries a single rRNA operon), albeit at rates 10-20% slower than wild-type ribosomes. These mutant Δ7 prrn strains accumulate free 30S particles and precursor 17S rRNA, indicative of biogenesis defects. Analysis of purified control and mutant subunits suggests that hALT stabilizes the inactive state by 1.2 kcal/mol with little-to-no impact on the active state or the transition state of conversion.

Ribosomes of Bacteroidia fail to recognize Shine–Dalgarno (SD) sequences due to sequestration of the 3′ tail of the 16S rRNA on the 30S platform. Yet in these organisms, theprfBgene typically contains the programmed +1 frameshift site with its characteristic SD sequence. Here, we investigateprfBautoregulation inFlavobacterium johnsoniae, a member of the Bacteroidia. We find that the efficiency ofprfBframeshifting inF. johnsoniaeis low (∼7%) relative to that inEscherichia coli(∼50%). Mutation or truncation of bS21 inF. johnsoniaeincreases frameshifting substantially, suggesting that anti-SD (ASD) sequestration is responsible for the reduced efficiency. The frameshift site of certain Flavobacteriales, such asWinogradskyella psychrotolerans, has no SD. InF. johnsoniae, thisW. psychrotoleranssequence supports frameshifting as well as the native sequence, and mutation of bS21 causes no enhancement. These data suggest thatprfBframeshifting normally occurs without SD–ASD pairing, at least under optimal laboratory growth conditions. Chromosomal mutations that remove the frameshift or ablate the SD confer subtle growth defects in the presence of paraquat or streptomycin, respectively, indicating that both the autoregulatory mechanism and the SD element contribute toF. johnsoniaecell fitness. Analysis ofprfBframeshift sites across 2686 representative bacteria shows loss of the SD sequence in many clades, with no obvious relationship to genome-wide SD usage. These data reveal unexpected variation in the mechanism of frameshifting and identify another group of organisms, the Verrucomicrobiales, that globally lack SD sequences.

Ribosome biogenesis occurs co-transcriptionally and entails rRNA folding, ribosomal 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.

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 rpsU mRNA 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.