Crystal Structure of an Initiation Factor Bound to the 30
            <i>S</i>
            Ribosomal Subunit

Carter, Andrew P.; Clemons, William M.; Brodersen, D.E.; Morgan-Warren, Robert J.; Hartsch, Thomas; Wimberly, Brian T.; Ramakrishnan, V.

doi:10.1126/science.1057766

Cited by 357 publications

(415 citation statements)

References 38 publications

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“…Upon RRF binding, the tip of the helix 44 goes through a rotation (Ϸ18°) around the axis of the helix and a shift (Ϸ7 Å, Fig. 2c), which are of similar magnitudes as reported previously upon binding of EF-G (26,27) and IF1 (2). The position of the tip of 23S rRNA helix 69 in RRF-bound and unbound maps are significantly different (Fig.…”

Section: Resultssupporting

confidence: 84%

“…Translation on the ribosome comprises of four main steps: (i) initiation, (ii) elongation, (iii) termination, and (iv) recycling. Recent advancements in structural studies of the translational machinery have helped elucidate binding positions and functions of various translational factors involved in various stages of initiation (1,2), elongation (3,4), and termination (5-7). The fourth step of translation requires binding of a dedicated protein factor, the ribosome-recycling factor (RRF), which in conjunction with elongation factor G (EF-G) helps removing the mRNA and last deacylated tRNA from the ribosome (see ref.…”

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confidence: 99%

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Visualization of ribosome-recycling factor on the Escherichia coli 70S ribosome: Functional implications

Agrawal

Sharma

Kiel

et al. 2004

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

148

170

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After the termination step of protein synthesis, a deacylated tRNA and mRNA remain associated with the ribosome. The ribosomerecycling factor (RRF), together with elongation factor G (EF-G), disassembles this posttermination complex into mRNA, tRNA, and the ribosome. We have obtained a three-dimensional cryo-electron microscopic map of a complex of the Escherichia coli 70S ribosome and RRF. We find that RRF interacts mainly with the segments of the large ribosomal subunit's (50S) rRNA helices that are involved in the formation of two central intersubunit bridges, B2a and B3. The binding of RRF induces considerable conformational changes in some of the functional domains of the ribosome. As compared to its binding position derived previously by hydroxyl radical probing study, we find that RRF binds further inside the intersubunit space of the ribosome such that the tip of its domain I is shifted (by Ϸ13 Å) toward protein L5 within the central protuberance of the 50S subunit, and domain II is oriented more toward the small ribosomal subunit (30S). Overlapping binding sites of RRF, EF-G, and the P-site tRNA suggest that the binding of EF-G would trigger the removal of deacylated tRNA from the P site by moving RRF toward the ribosomal E site, and subsequent removal of mRNA may be induced by a shift in the position of 16S rRNA helix 44, which harbors part of the mRNA. R ibosomes are responsible for translating genetic information carried by mRNAs into specific sequences of amino acids. Translation on the ribosome comprises of four main steps: (i) initiation, (ii) elongation, (iii) termination, and (iv) recycling. Recent advancements in structural studies of the translational machinery have helped elucidate binding positions and functions of various translational factors involved in various stages of initiation (1, 2), elongation (3, 4), and termination (5-7). The fourth step of translation requires binding of a dedicated protein factor, the ribosome-recycling factor (RRF), which in conjunction with elongation factor G (EF-G) helps removing the mRNA and last deacylated tRNA from the ribosome (see ref. 8).Atomic structures of RRF determined from five different species, including Escherichia coli, show that it is comprised of two structural domains: domain I, consisting of three long ␣-helix bundles, and the smaller domain II, which is an ␣͞␤ motif (9-13). Different orientations of domain II in these structures have been attributed to interdomain flexibility, which is thought to be necessary for RRF to function on the ribosome (12).The overall match in dimensions between RRF and tRNA (9) prompted the proposal of structural and functional molecular mimicry between the two molecules (9). In a recent study (14) using the hydroxyl radical probing (HRP) method, the orientation of RRF on the ribosome was derived. This study did not support the idea of direct molecular mimicry of tRNA by RRF, because the derived binding position of RRF was quite different from that one would expect based on structural mimicry. The inferred bin...

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Section: Resultssupporting

confidence: 84%

mentioning

confidence: 99%

Visualization of ribosome-recycling factor on the Escherichia coli 70S ribosome: Functional implications

Agrawal

Sharma

Kiel

et al. 2004

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

148

170

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“…First, in structural models, the A site is seen to be exposed on the surface of the small 30 S subunit [28]. Secondly, unpaired base residues A 1492 and A 1493 within the A site are crucial to the decoding process and to codon/anticodon recognition [4], the selection of tRNA by the ribosome [29], as well as to the binding of initiation factor 1 [30]. Thirdly, the A site is well known to be the site of interaction of aminoglycoside antibiotics [4,31], which can therefore provide tools for assay development and target validation.…”

Section: Resultsmentioning

confidence: 99%

Targeting the A site RNA of the Escherichia coli ribosomal 30 S subunit by 2′-O-methyl oligoribonucleotides: a quantitative equilibrium dialysis binding assay and differential effects of aminoglycoside antibiotics

Abelian

Walsh

Lentzen

et al. 2004

Biochemical Journal

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The bacterial ribosome comprises 30 S and 50 S ribonucleoprotein subunits, contains a number of binding sites for known antibiotics and is an attractive target for selection of novel antibacterial agents. On the 30 S subunit, for example, the A site (aminoacyl site) close to the 3′-end of 16 S rRNA is highly important in the decoding process. Binding by some aminoglycoside antibiotics to the A site leads to erroneous protein synthesis and is lethal for bacteria. We targeted the A site on purified 30 S ribosomal subunits from Escherichia coli with a set of overlapping, complementary OMe (2′-O-methyl) 10-mer oligoribonucleotides. An equilibrium dialysis technique was applied to measure dissociation constants of these oligonucleotides. We show that there is a single high-affinity region, spanning from A1493 to C1510 (Kd, 29–130 nM), flanked by two lower-affinity regions, within a span from U1485 to G1516 (Kd, 310–4300 nM). Unexpectedly, addition of the aminoglycoside antibiotic paromomycin (but not hygromycin B) caused a dose-dependent increase of up to 7.5-fold in the binding of the highest affinity 10-mer 1493 to 30 S subunits. Oligonucleotides containing residues complementary to A1492 and/or A1493 showed particularly marked stimulation of binding by paromomycin. The results are consistent with high-resolution structures of antibiotic binding to the A site and with greater accessibility of residues of A1492 and A1493 upon paromomycin binding. 10-mer 1493 binding is thus a probe of the conformational switch to the ‘closed’ conformation triggered by paromomycin that is implicated in the discrimination by 30 S subunits of cognate from non-cognate tRNA and the translational misreading caused by paromomycin. Finally, we show that OMe oligonucleotides targeted to the A site are moderately good inhibitors of in vitro translation and that there is a limited correlation of inhibition activity with binding strength to the A site.

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“…It was therefore suggested that IF1 mimics the A-site-bound tRNA and could prevent premature binding of aminoacyl-tRNA by blocking the A-site on the 30S ribosomal subunit [16]. The crystal structure of IF1 bound to the 30S subunit has shown that IF1 indeed occludes the ribosomal A-site and flips out the functionally important bases A1492 and A1493 from helix 44 of 16S RNA, hiding these bases inside IF1 [17]. These latter X-ray data suggest that IF1 binds to the A-site in a different manner than does the A-site-bound tRNA [16,18,19].…”

Section: Introductionmentioning

confidence: 99%

In vivo involvement of mutated initiation factor IF1 in gene expression control at the translational level

2005

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Edited by Lev KisselevAbstract The influence in vivo of mutated forms of translation initiation factor (IF1) on the expression of the lacZ or 3A 0 reporter genes, with different initiation and/or +2 codons, has been investigated. Reporter gene expression in these infA(IF1) mutants is similar to the wild-type strain. The results do not support the longstanding hypothesis that IF1 could perform discriminatory functions while blocking the aminoacyl-tRNA acceptor site (A-site) of the ribosome. One cold-sensitive IF1 mutant shows a general overexpression, in particular at low temperatures, of both reporter genes at the protein but not mRNA level.

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Crystal Structure of an Initiation Factor Bound to the 30 S Ribosomal Subunit

Cited by 357 publications

References 38 publications

Visualization of ribosome-recycling factor on the Escherichia coli 70S ribosome: Functional implications

Visualization of ribosome-recycling factor on the Escherichia coli 70S ribosome: Functional implications

Targeting the A site RNA of the Escherichia coli ribosomal 30 S subunit by 2′-O-methyl oligoribonucleotides: a quantitative equilibrium dialysis binding assay and differential effects of aminoglycoside antibiotics

In vivo involvement of mutated initiation factor IF1 in gene expression control at the translational level

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