SummaryTranslational control is widely used to adjust gene expression levels. During the stringent response in bacteria, mRNA is degraded on the ribosome by the ribosome-dependent endonuclease, RelE. The molecular basis for recognition of the ribosome and mRNA by RelE and the mechanism of cleavage are unknown. Here, we present crystal structures of E. coli RelE in isolation (2.5 Å) and bound to programmed Thermus thermophilus 70S ribosomes before (3.3 Å) and after (3.6 Å) cleavage. RelE occupies the A site and causes cleavage of mRNA after the second nucleotide of the codon by reorienting and activating the mRNA for 2′-OH-induced hydrolysis. Stacking of A site codon bases with conserved residues in RelE and 16S rRNA explains the requirement for the ribosome in catalysis and the subtle sequence specificity of the reaction. These structures provide detailed insight into the translational regulation on the bacterial ribosome by mRNA cleavage.
Mtr4 is a conserved RNA helicase that functions together with the nuclear exosome. It participates in the processing of structured RNAs, including the maturation of 5.8S ribosomal RNA (rRNA). It also interacts with the polyadenylating Trf4-Air2 heterodimer to form the so-called TRAMP (Trf4-Air2-Mtr4 Polyadenylation) complex. TRAMP is involved in exosome-mediated degradation of aberrant RNAs in nuclear surveillance pathways. We report the 2.9-A resolution crystal structure of Saccharomyces cerevisiae Mtr4 in complex with ADP and RNA. The structure shows a central ATPase core similar to that of other DExH helicases. Inserted in the DExH core is a region characteristic of Mtr4 orthologues that folds into an elongated stalk connected to a beta-barrel domain. This domain shows unexpected similarity to the KOW domain of L24, a ribosomal protein that binds 23S rRNA. We find that indeed the KOW domain of Mtr4 is able to bind in vitro transcribed tRNA(iMet), suggesting it might assist in presenting RNA substrates to the helicase core. The interaction of Mtr4 with Trf4-Air2 is mediated not by the stalk/KOW insertion but by the DExH core. We find that in the context of the TRAMP complex, the DExH core functions independently in vitro as an RNA helicase and a protein-binding platform. Mtr4 has thus evolved specific structural and surface features to perform its multiple functions.
The ribosome carries out the synthesis of proteins in every living cell. It consequently represents a frontline target in anti-microbial therapy. Tuberculosis ranks among the leading causes of death worldwide, due in large part to the combination of difficult-to-treat latency and antibiotic resistance. Here, we present the 3.3-Å cryo-EM structure of the 70S ribosome of Mycobacterium smegmatis, a close relative to the human pathogen Mycobacterium tuberculosis. The structure reveals two additional ribosomal proteins and localizes them to the vicinity of drug-target sites in both the catalytic center and the decoding site of the ribosome. Furthermore, we visualized actinobacterium-specific rRNA and protein expansions that extensively remodel the ribosomal surface with implications for polysome organization. Our results provide a foundation for understanding the idiosyncrasies of mycobacterial translation and reveal atomic details of the structure that will facilitate the design of anti-tubercular therapeutics.
The TRAMP complex is involved in the nuclear surveillance and turnover of noncoding RNAs and intergenic transcripts. TRAMP is associated with the nuclear exosome and consists of a poly(A)polymerase subcomplex (Trf4-Air2) and a helicase (Mtr4). We found that N-terminal low-complexity regions of Trf4 and Air2 bind Mtr4 in a cooperative manner. The 2.4 Å resolution crystal structure of the corresponding ternary complex reveals how Trf4 and Air2 wrap around the DExH core of the helicase. Structure-based mutations on the DExH core impair binding to Trf4 and Air2, and also to Trf5 and Air1. The combination of structural, biochemical, and biophysical data suggests that the poly(A)polymerase core of Trf4-Air2 is positioned below the base of the helicase, where the unwound 3' end of an RNA substrate is expected to emerge. The results reveal conceptual similarities between the two major regulators of the exosome, the nuclear TRAMP and cytoplasmic Ski complexes.
Ribosome-associated complex (RAC) consists of the Hsp40 homolog Zuo1 and the Hsp70 homolog Ssz1. The chaperone participates in the biogenesis of newly synthesized polypeptides. Here we have identified yeast Rpl31, a component of the large ribosomal subunit, as a contact point of RAC at the polypeptide tunnel exit. Rpl31 is encoded by RPL31a and RPL31b, two closely related genes. ⌬rpl31a⌬rpl31b displayed slow growth and sensitivity to low as well as high temperatures. In addition, ⌬rpl31a⌬rpl31b was highly sensitive toward aminoglycoside antibiotics and suffered from defects in translational fidelity. With the exception of sensitivity at elevated temperature, the phenotype resembled yeast strains lacking one of the RAC subunits or Rpl39, another protein localized at the tunnel exit. Defects of ⌬rpl31a⌬rpl31b⌬zuo1 did not exceed that of ⌬rpl31a⌬rpl31b or ⌬zuo1. However, the combined deletion of RPL31a, RPL31b, and RPL39 was lethal. Moreover, RPL39 was a multicopy suppressor, whereas overexpression of RAC failed to rescue growth defects of ⌬rpl31a⌬rpl31b. The findings are consistent with a model in that Rpl31 and Rpl39 independently affect a common ribosome function, whereas Rpl31 and RAC are functionally interdependent. Rpl31, while not essential for binding of RAC to the ribosome, might be involved in proper function of the chaperone complex. INTRODUCTIONTwo Hsp70 family members Ssb1/2 (Ssb1 and Ssb2 differ by only four amino acids) and Ssz1 and one J-domain protein (Zuo1) are abundant components of the translation machinery of Saccharomyces cerevisiae (Raue et al., 2007). The three chaperones are genetically linked and form a functional triad. Lack of either SSB1/2, SSZ1, or ZUO1 results in slow growth, cold sensitivity, and pronounced hypersensitivity against aminoglycosides such as paromomycin (Gautschi et al., 2002;Hundley et al., 2002). Ssz1 and Zuo1 assemble into a stable heterodimeric complex termed RAC (ribosome-associated complex). RAC acts as a cochaperone for Ssb1/2 and stimulates its ATP hydrolysis. The function requires both RAC subunits (Huang et al., 2005;Conz et al., 2007).RAC is anchored to the ribosome via Zuo1 (Gautschi et al., 2001). The idea is that positioning of RAC on the ribosome is required for its interaction with Ssb1/2 (Yan et al., 1998). However, the function of Ssz1 does not strictly depend on stable interaction with Zuo1 or ribosomes (Conz et al., 2007). How exactly Zuo1 anchors RAC is currently unclear. It was proposed that Zuo1 binds to ribosomes, in part, by interaction with rRNA (Yan et al., 1998). However, purified Zuo1 unspecifically interacts with a variety of nucleic acids. Initially, Zuo1 was identified via its ability to interact with Z-DNA (Zhang et al., 1992), it also interacts tightly with tRNA (Wilhelm et al., 1994) and recently was shown to bind to a small inhibitor RNA (Raychaudhuri et al., 2006). The mouse homolog MIDA1 interacts with DNA that forms small stem loop structures (Inoue et al., 2000). The diversity of nucleic acids that interact with Zuo1 raises the qu...
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