The RNA‐binding protein Hfq is important for ribosome biogenesis and affects translation fidelity

Ribosome biogenesis is a complex process involving multiple factors. Here, we show that the widely conserved RNA chaperone Hfq, which can regulate sRNA‐mRNA basepairing, plays a critical role in rRNA processing and ribosome assembly in Escherichia coli. Hfq binds the 17S rRNA precursor and facilitates its correct processing and folding to mature 16S rRNA. Hfq assists ribosome assembly and associates with pre‐30S particles but not with mature 30S subunits. Inactivation of Hfq strikingly decreases the pool of mature 70S ribosomes. The reduction in ribosome levels depends on residues located in the distal face of Hfq but not on residues found in the proximal and rim surfaces which govern interactions with the sRNAs. Our results indicate that Hfq‐mediated regulation of ribosomes is independent of its function as sRNA‐regulator. Furthermore, we observed that inactivation of Hfq compromises translation efficiency and fidelity, both features of aberrantly assembled ribosomes. Our work expands the functions of the Sm‐like protein Hfq beyond its function in small RNA‐mediated regulation and unveils a novel role of Hfq as crucial in ribosome biogenesis and translation.     

Capacity for RNA synthesis in 70S ribosome-deficient plastids of heat-bleached rye leaves

In the leaves of rye seedlings (Secale cereale L.) grown at an elevated temperature of 32°C the formation of plastidic 70S ribosomes is specifically prevented. The resulting plastid ribosome-deficient leaves, which are chlorotic in light, represent a system for the identification of translation products of the 80S ribosomes among the chloroplastic proteins. Searching for the primary heat-sensitive event causing the 70S ribosome-deficiency, the thermostability of the chloroplastic capacity for RNA synthesis was investigated. The RNA polymerase activity of isolated normal chloroplasts from 22°-grown rye leaves was not inactivated in vitro at temperatures between 30° and 40°C. The ribosome-deficient plastids purified from bleached 32°-grown leaf parts contained significant RNA polymerase activity which was, however, lower than in functional chloroplasts. After application of [³H]uridine to intact leaf tissues [³H]uridine incorporation was found in ribosome-deficient plastids of 32°C-grown leaves. The amount of incorporation was similar to that in the control chloroplasts from 22°C-grown leaves. According to these results, it is unlikely that the non-permissive temperature (32°C) causes a general inactivation of the chloroplastic RNA synthesis in rye leaves.     

Translation initiation with 70S ribosomes: an alternative pathway for leaderless mRNAs

It is generally accepted that translation in bacteria is initiated by 30S ribosomal subunits. In contrast, several lines of rather indirect in vitro evidence suggest that 70S monosomes are capable of initiating translation of leaderless mRNAs, starting with the A of the initiation codon. In this study, we demonstrate the proficiency of dedicated 70S ribosomes in in vitro translation of leaderless mRNAs. In support, we show that a natural leaderless mRNA can be translated with crosslinked 70S wild-type ribosomes. Moreover, we report that leaderless mRNA translation continues under conditions where the prevalence of 70S ribosomes is created in vivo, and where translation of bulk mRNA ceases. These studies provide in vivo as well as direct in vitro evidence for a 70S initiation pathway of a naturally occurring leaderless mRNA, and are discussed in light of their significance for bacterial growth under adverse conditions and their evolutionary implications for translation.     

Role of polyamines in the binding of initiator tRNA to the 70S ribosomes of extreme thermophilic bacterium Calderobacterium hydrogenophilum

Slowly cooled cells of an extreme thermophilic eubacterium Calderobacterium hydrogenophilum possess ribosomes with weakly associated subunits. These ribosomal subunits are capable of association to 70S ribosomes either at higher Mg²⁺ concentrations (30–40 mM) or at 4–10 mM Mg²⁺ and in the presence of polyamines. The contribution of 30S and 50S subunits to the hydrodynamic stability of ribosomes was examined by forming hybrid 30S–50S couples from C. hydrogenophilum and Escherichia coli. At lower Mg²⁺ (4–10 mM) heterogeneous subunits containing 30S E. coli and 50S C. hydrogenophilum and homogeneous subunits of the thermophilic bacterium associated only in the presence of polyamines. Ribosomal subunits associated at 30 mM Mg²⁺ lose thermal stability and activity concerning poly(AUG)-dependent binding of f[³H]Met-tRNA to the P-site on 70S ribosomes or translation of poly(UG). Poly(AUG), deacylated-tRNA or initiator-tRNA have no valuable effect on association of 30S and 50S subunits. Protein synthesis initiation factor IF3 of C. hydrogenophilum prevents association of ribosomal subunits to 70S ribosomes at physiological temperature (70°C). The factor also stimulates dissociation of 70S ribosomes of E. coli at 37°C. The codon-specific binding of f[³H]Met-tRNA to homogeneous 70S ribosomes of C. hydrogenophilum at 70°C is dependent on the presence of initiation factors and concentrations of tri-pentaamines. However, excess of polyamines inhibited the reaction. Our results indicate that tri-pentaamines enhance conformational stability of 70S initiation complex at elevated temperatures.     

The Hfq chaperone helps the ribosome mature

Ribosome biogenesis is a complex process that is facilitated by a large number of assembly factors. In this issue, Andrade et al ( 2018 ) provide evidence that a widely conserved RNA chaperone, Hfq, acts as a ribosomal assembly factor in bacteria. Hfq is known to support regulation of stress response genes by small RNAs. Andrade et al ( 2018 ) show that the absence of Hfq results in higher levels of immature 30S ribosomes and error‐prone translation, suggesting that Hfq globally affects the quality of protein synthesis when bacteria are under stress.     

Protection of specific sites in 23 S and 5 S RNA from chemical modification by association of 30 S and 50 S ribosomes.

The effect of 30S subunit attachment on the accessibility of specific sites in 5 S and 23 S RNA in 50 S ribosomal subunits was studied by means of the guanine-specific reagent kethoxal. Oligonucleotides surrounding the sites of kethoxal substitution were resolved and quantitated by diagonal electrophoresis. In contrast to the extensive protection of sites in 16 S RNA in 70 S ribosomes (Chapman &; Noller, 1977), only two strongly (approx. 90%) protected sites were detected in 23 S RNA. The nucleotide sequences at these sites are

in which the indicated kethoxal-reactive guanines (with K above them) are strongly protected by association of 30 S and 50 S subunits. The latter sequence has the potential to base-pair with nucleotides 816 to 821 of the 16 S RNA, a site which has been shown to be protected from kethoxal by 50 S subunits and essential for subunit association. Six additional sites in 23 S RNA are partially (30 to 50%) protected by 30 S subunits. One of these sequences,

is complementary to nucleotides 787 to 792 of 16 S RNA. a site which is also 50 S-protected and essential for association. Of the two kethoxal-reactive 5 S RNA sites in 50 S subunits, G₁₃ is partially protected in 70 S ribosomes. while G₄₁ remains unaffected by subunit association.The relatively small number of kethoxal-reactive sites in 23 S RNA that is strongly protected in 70 S ribosomes suggests that subunit association may involve contacts between single-stranded sites in 16 S RNA and 50 S subunit proteins or non-Watson-Crick interactions with 23 S RNA. in addition to the two suggested base-paired contacts.     

Small protein B interacts with the large and the small subunits of a stalled ribosome during trans-translation

During trans-translation, stalled bacterial ribosomes are rescued by small protein B (SmpB) and by transfer-messenger RNA (tmRNA). Stalled ribosomes switch translation from the defective messages to a short internal reading frame on tmRNA that tags the nascent peptide chain for degradation and recycles the ribosomes. We present evidences that SmpB binds the large and small ribosomal subunits in vivo and in vitro. The binding between SmpB and the ribosomal subunits is very tight, with a dissociation constant of 1.7 × 10⁻¹⁰ M, similar to its K_D for the 70S ribosome or for tmRNA. tmRNA displaces SmpB from its 50S binding but not from the 30S. In vivo, SmpB is detected on the 50S when trans-translation is impaired by lacking tmRNA or a functional SmpB. SmpB contacts the large subunit transiently and early during the trans-translational process. The affinity of SmpB for the two ribosomal subunits is modulated by tmRNA in the course of trans-translation. It is the first example of two copies of the same protein interacting with two different functional sites of the ribosomes.     

Ribosome Binding Site of Qβ RNA Polymerase Cistron

IN conditions of polypeptide chain initiation, 70S ribosomes bind to intact bacteriophage RNA predominantly at the initiation region of the coat protein cistron^1,2. The binding sites of the A protein and RNA polymerase cistrons are fully available only after modification of the secondary or tertiary structure of the RNA^2,3.     

Recycling factors for ribosome disassembly in the apicoplast and mitochondrion of Plasmodium falciparum

The reduced genomes of the apicoplast and mitochondrion of the malaria parasite Plasmodium falciparum are actively translated and antibiotic‐mediated translation inhibition is detrimental to parasite survival. In order to understand recycling of organellar ribosomes, a critical step in protein translation, we identified ribosome recycling factors (RRF) encoded by the parasite nuclear genome. Targeting of PfRRF1 and PfRRF2 to the apicoplast and mitochondrion respectively was established by localization of leader sequence–GFP fusions. Unlike any RRF characterized thus far, PfRRF2 formed dimers with disulphide interaction(s) and additionally localized in the cytoplasm, thus suggesting adjunct functions for the factor. PfRRF1 carries a large 108‐amino‐acid insertion in the functionally critical hinge region between the head and tail domains of the protein, yet complemented Escherichia coli RRF in the LJ14frr^ts mutant and disassembled surrogate E. coli 70S ribosomes in the presence of apicoplast‐targeted EF‐G. Recombinant PfRRF2 bound E. coli ribosomes and could split monosomes in the presence of the relevant mitochondrial EF‐G but failed to complement the LJ14frr^ts mutant. Although proteins comprising subunits of P. falciparum organellar ribosomes are predicted to differ from bacterial and mitoribosomal counterparts, our results indicate that the essential interactions required for recycling are conserved in parasite organelles.