Insights into Translational Termination from the Structure of RF2 Bound to the Ribosome

Weixlbaumer, A.; Jin, Hong; Neubauer, Cajetan; Voorhees, R.M.; Petry, Sabine; Kelley, A.C.; Ramakrishnan, V.

doi:10.1126/science.1164840

Cited by 289 publications

(477 citation statements)

References 31 publications

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“…The interaction of A6 with the conserved C1054 provides one mechanism by which HigB selects for adenosines at the +6 position. HigB recognition of the +6 nucleotide is most similar to how release factors 1 or 2 (RF1/2) recognize stop codons, with one significant difference being the +6 nucleotide base stacks with decoding center nucleotide G530 upon RF binding, an interaction absent in HigB-mRNA recognition (41,42).…”

Section: Resultsmentioning

confidence: 94%

Defining the mRNA recognition signature of a bacterial toxin protein

Schureck

Dunkle

Maehigashi

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Bacteria contain multiple type II toxins that selectively degrade mRNAs bound to the ribosome to regulate translation and growth and facilitate survival during the stringent response. Ribosomedependent toxins recognize a variety of three-nucleotide codons within the aminoacyl (A) site, but how these endonucleases achieve substrate specificity remains poorly understood. Here, we identify the critical features for how the host inhibition of growth B (HigB) toxin recognizes each of the three A-site nucleotides for cleavage. X-ray crystal structures of HigB bound to two different codons on the ribosome illustrate how HigB uses a microbial RNase-like nucleotide recognition loop to recognize either cytosine or adenosine at the second A-site position. Strikingly, a single HigB residue and 16S rRNA residue C1054 form an adenosine-specific pocket at the third A-site nucleotide, in contrast to how tRNAs decode mRNA. Our results demonstrate that the most important determinant for mRNA cleavage by ribosome-dependent toxins is interaction with the third A-site nucleotide. (1,3,4). This rapid inhibitory switch suppresses metabolite consumption and temporarily halts cell growth to promote bacterial survival until nutrients are readily available. Among the prosurvival genes regulated by (p)ppGpp production are toxin-antitoxin modules, which have additional roles in antibiotic resistance and tolerance, biofilm and persister cell formation, and niche-specific colonization (5-11). The critical roles toxin-antitoxin pairs play in bacterial physiology underscore the importance of understanding their molecular targets and modes of action.There are five different classes (I to V) of toxin-antitoxin systems defined by how the antitoxin represses toxin function (1). Type II toxin-antitoxin pairs form protein-protein complexes during exponential growth that serve two purposes: inhibition of toxin activity by antitoxin binding and transcriptional autorepression to limit toxin expression (12). Antitoxins are proteolytically degraded after (p)ppGpp accumulation, leading to derepression at the toxin-antitoxin promoter (8, 12). Liberated toxin proteins inhibit the replication or translation machinery by targeting DNA gyrase, initiator tRNA fMet , glutamyl-tRNA synthetase, EF-Tu, free mRNA, ribosome-bound mRNA, and the ribosome itself (13-20).Ribosome-dependent toxins cleave mRNAs on the ribosome between the second and third nucleotides of the aminoacyl (A)-site codon (21-23). Although collectively Escherichia coli ribosome-dependent toxins target a diverse range of codons, each individual toxin appears to have a strong codon preference and cleaves at defined positions along the mRNA (24-26). RelE cleaves at UAG stop codons and the CAG sense codon (all codons denoted in the 5′-3′ direction); YoeB cleaves at codons following a translational AUG start site and at the UAA stop codon; and YafQ cleaves a single AAA sense codon (16,24,(27)(28)(29). In contrast, Proteus vulgaris host inhibition of growth B (HigB) toxin degrades multiple codons encod...

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Section: Resultsmentioning

confidence: 94%

Defining the mRNA recognition signature of a bacterial toxin protein

Schureck

Dunkle

Maehigashi

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…The specificity of the defects, observed only with RF2-UGA interactions, can perhaps be attributed to subtle differences observed on comparing the switch loop regions of RF1 and RF2 (34). For example, RF2 is seen to make additional contacts with H69 residues 1914 and 1915 (8), and these might be somewhat destabilized on interaction with the UGA-programmed ribosome complex. Whatever the cause, the specific deficiencies that we observe in the G1922A ribosomes are consistent with multiple earlier reports of RF-related deficiencies with H69 variant ribosomes (14 -16).…”

Section: Discussionmentioning

confidence: 99%

Helix 69 Is Key for Uniformity during Substrate Selection on the Ribosome

Ortiz‐Meoz

Green

2011

Journal of Biological Chemistry

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Structural studies of ribosome complexes with bound tRNAs and release factors show considerable contacts between these factors and helix 69 (H69) of 23 S rRNA. Although biochemical and genetic studies have provided some general insights into the role of H69 in tRNA and RF selection, a detailed understanding of these contributions remains elusive. Here, we present a presteady-state kinetic analysis establishing that two distinct regions of H69 make critical contributions to substrate selection. The loop of H69 (A1913) forms contacts necessary for the efficient accommodation of a subset of natural tRNA species, whereas the base of the stem (G1922) is specifically critical for UGA codon recognition by the class 1 release factor RF2. These data define a broad and critical role for this centrally located intersubunit helix (H69) in accurate and efficient substrate recognition by the ribosome.The ribosome is the ribonucleoprotein machine responsible for the faithful translation of the genetic material into the encoded polypeptide. The core RNA components of the ribosome (the 16 S and 23 S rRNA) that reflect its earliest evolution play a key role in interactions that specify the selection of tRNAs and release factors on recognition of sense and stop codons, respectively. During translation elongation, the selection of a cognate ternary complex (composed of aa-tRNA, EFTu, and GTP) from solution is specified primarily in the small ribosomal subunit where three nucleotides in 16 S rRNA directly evaluate the geometry of the codon-anticodon interaction (1). Though different in molecular detail, the selection of class 1 release factors takes place in the same decoding center of the small ribosomal subunit where conserved protein features of the RF engage the stop codon. Thus, for both decoding events, molecular interactions in the small subunit "decoding center" are early and critical contributors to the selection of cognate substrates during the translational cycle.In addition to these interactions in the decoding center, it is believed that other molecular interactions between the ribosome and these substrates contribute to binding and specificity. Structural studies of tRNA (2-5), release factors (6 -8), and ribosome recycling factor (9, 10) bound to the ribosome reveal that H69 2 of the large subunit rRNA makes extensive contacts with all of these factors. This universally conserved helix forms an intersubunit bridge that is proximal to the substrate binding cleft and directly contacts the functionally critical h44 of 16 S rRNA which contains two of the three nucleotides known to directly interact with the decoding helix (codon-anticodon) during tRNA selection (A1492 and A1493).What is the specific nature of these molecular interactions, and what do they suggest about the role of H69 in ribosome function? H69 contacts incoming tRNA in both the pre-and postaccommodation steps near positions 25/26 of the D stem and position 38, located near the anticodon stem; tRNA mutations at these positions are linked to miscoding pheno...

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“…These key conserved nucleotides from the decoding site of the small subunit undergo substantial rearrangements in response to the pairing of cognate codons and anticodons, 43 binding of IF1, 44 binding of antibiotics 43 or, to a less extent, recognition of a stop codon by release factors RF1 or RF2. [45][46][47] However, in spite of the observed reactivity changes, mutations at these positions do not reduce SmpB binding to the decoding site 42 or reduce the rate of peptidyl-transfer onto tmRNA (A. Buskirk, personal communication). Therefore, other contacts between SmpB and the decoding site that are different from those induced during translation elongation, are likely to take place with the surrounding nucleotides of the 16S RNA since positively charged SmpB has potential for interacting with negatively charged rRNA.…”

Section: O N O T D I S T R I B U T Ementioning

confidence: 99%