Peptidyl transferase center activity observed in single ribosomes

Sytnik, Alexander; Vladimirov, S.N.; Jia, Yiwei; Li, Liangquan; Cooperman, Barry S.; Hochstrasser, Robin M.

doi:10.1006/jmbi.1998.2312

Cited by 27 publications

(14 citation statements)

References 32 publications

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“…Key L2-S1 tertiary structural interactions can be envisioned to function as kinetic barriers to the unfolding of pseudoknot-forming stem S2, in the same way that Mg 2ϩ -dependent tertiary structural interactions function as kinetic barriers to the unwinding of component helices in larger RNAs (51). The related ScYLV and BWYV pseudoknotted RNAs characterized here constitute an excellent model system with which to further address mechanistic (52,53) and kinetic (54) issues relevant for understanding mRNA pseudoknot-stimulated Ϫ1 PRF by structurally very similar mRNAs.…”

Section: Discussionmentioning

confidence: 97%

A loop 2 cytidine-stem 1 minor groove interaction as a positive determinant for pseudoknot-stimulated –1 ribosomal frameshifting

Cornish

Hennig

Giedroc

2005

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

170

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The molecular determinants of stimulation of ؊1 programmed ribosomal frameshifting (؊1 PRF) by RNA pseudoknots are poorly understood. Sugarcane yellow leaf virus (ScYLV) encodes a 28-nt mRNA pseudoknot that promotes ؊1 PRF between the P1 (protease) and P2 (polymerase) genes in plant luteoviruses. The solution structure of the ScYLV pseudoknot reveals a well ordered loop 2 (L2) that exhibits continuous stacking of A20 through C27 in the minor groove of the upper stem 1 (S1), with C25 flipped out of the triple-stranded stack. Five consecutive triple base pairs flank the helical junction where the 3 nucleotide of L2, C27, adopts a cytidine 27 N3-cytidine 14 2 -OH hydrogen bonding interaction with the C14-G7 base pair. This interaction is isosteric with the adenosine N1-2 -OH interaction in the related mRNA from beet western yellows virus (BWYV); however, the ScYLV and BWYV mRNA structures differ in their detailed L2-S1 hydrogen bonding and L2 stacking interactions. Functional analyses of ScYLV͞BWYV chimeric pseudoknots reveal that the ScYLV RNA stimulates a higher level of ؊1 PRF (15 ؎ 2%) relative to the BWYV pseudoknot (6 ؎ 1%), a difference traced largely to the identity of the 3 nucleotide of L2 (C27 vs. A25 in BWYV). Strikingly, C27A ScYLV RNA is a poor frameshift stimulator (2.0%) and is destabilized by Ϸ1.5 kcal⅐mol ؊1 (pH 7.0, 37°C) with respect to the wild-type pseudoknot. These studies establish that the precise network of weak interactions nearest the helical junction in structurally similar pseudoknots make an important contribution to setting the frameshift efficiency in mRNAs.base triple ͉ RNA pseudoknot ͉ translational recoding

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Section: Discussionmentioning

confidence: 97%

A loop 2 cytidine-stem 1 minor groove interaction as a positive determinant for pseudoknot-stimulated –1 ribosomal frameshifting

Cornish

Hennig

Giedroc

2005

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

170

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“…Such efforts necessitate immobilizing one or more components of the translation apparatus to detect translation reactions over extended periods. The first single-molecule studies of ribosome function focused on detecting PTC activity using puromycin (Sytnik et al, 1999). Subsequent advances in CCD technologies, microfluidic devices and fluorophore photophysics have since allowed the tracking of complete protein synthesis reactions (see, for example, Uemura et al, 2010; Wen et al, 2008).…”

Section: Antibiotics Modulate the Energy Landscape Of Ribosome Dynamicsmentioning

confidence: 99%

Probing Translation with Small-Molecule Inhibitors

Blanchard

Cooperman

Wilson

2010

Chemistry & Biology

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Summary The translational apparatus of the bacterial cell remains one of the principal targets of antibiotics for the clinical treatment of infection worldwide. Since the introduction of specific translation inhibitors into clinical practise in the late 1940’s, intense efforts have been made to understand their precise mechanisms of action. Such research has often revealed significant and sometimes unexpected insights into many fundamental aspects of the translation mechanism. Central to progress in this area, high-resolution crystal structures of the bacterial ribosome identifying the sites of antibiotic binding are now available, which, together with recent developments in single-molecule and fast-kinetic approaches, provide an integrated view of the dynamic translation process. Assays employing these approaches and focusing on specific steps of the overall translation process are amenable for drug-screening. Such assays, coupled with structural studies, have the potential not only to accelerate the discovery of novel and effective antimicrobial agents, but also to refine our understanding of the translation mechanism, since antibiotics often stabilize specific functional states of the ribosome and allow distinct translation steps to be dissected in molecular detail.

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“…It has been shown previously that ribosomes adsorbed on a mica surface are competent to bind aminoacyl-tRNA in the peptidyl transfer site (P-site) and to form a peptide bond with added puromycin (Sytnik et al 1999). Here we extend this work by measuring bulk poly(Phe) synthesis on poly(U)-programmed mica-bound ribosomes and detecting translocation of individual ribosome-bound poly(U) templates using optical microscopy to examine mRNA-tethered beads.…”

Section: Introductionmentioning

confidence: 99%

Protein synthesis by single ribosomes

Vanzi¹,

Vladimirov²,

Knudsen³

et al. 2003

RNA

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The ribosome is universally responsible for synthesizing proteins by translating the genetic code transcribed in mRNA into an amino acid sequence. Ribosomes use cellular accessory proteins, soluble transfer RNAs, and metabolic energy to accomplish the initiation, elongation, and termination of peptide synthesis. In translocating processively along the mRNA template during the elongation cycle, ribosomes act as supramolecular motors. Here we demonstrate that ribosomes adsorbed on a surface, as for mechanical or spectroscopic studies, are capable of polypeptide synthesis and that tethered particle analysis of fluorescent beads connected to ribosomes via polyuridylic acid can be used to estimate the rate of polyphenylalanine synthesis by individual ribosomes. This work opens the way for application of biophysical techniques, originally developed for the classical motor proteins, to the understanding of protein biosynthesis.

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Peptidyl transferase center activity observed in single ribosomes

Cited by 27 publications

References 32 publications

A loop 2 cytidine-stem 1 minor groove interaction as a positive determinant for pseudoknot-stimulated –1 ribosomal frameshifting

A loop 2 cytidine-stem 1 minor groove interaction as a positive determinant for pseudoknot-stimulated –1 ribosomal frameshifting

Probing Translation with Small-Molecule Inhibitors

Protein synthesis by single ribosomes

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