Rachel Green | University of California, Santa Barbara (original) (raw)

Papers by Rachel Green

Nature, 2009

The overall fidelity of protein synthesis has been thought to rely on the combined accuracy of tw... more The overall fidelity of protein synthesis has been thought to rely on the combined accuracy of two basic processes: the aminoacylation of transfer RNAs with their cognate amino acid by the aminoacyl-tRNA synthetases, and the selection of cognate aminoacyl-tRNAs by the ribosome in cooperation with the GTPase elongation factor EF-Tu. These two processes, which together ensure the specific acceptance of a correctly charged cognate tRNA into the aminoacyl (A) site, operate before peptide bond formation. Here we report the identification of an additional mechanism that contributes to high fidelity protein synthesis after peptidyl transfer, using a well-defined in vitro bacterial translation system. In this retrospective quality control step, the incorporation of an amino acid from a non-cognate tRNA into the growing polypeptide chain leads to a general loss of specificity in the A site of the ribosome, and thus to a propagation of errors that results in abortive termination of protein synthesis.

Cell, 2009

The faithful and rapid translation of genetic information into peptide sequences is an indispensa... more The faithful and rapid translation of genetic information into peptide sequences is an indispensable property of the ribosome. The mechanistic understanding of strategies used by the ribosome to achieve both speed and fidelity during translation results from nearly a half century of biochemical and structural studies. Emerging from these studies is the common theme that the ribosome uses local as well as remote conformational switches to govern induced-fit mechanisms that ensure accuracy in codon recognition during both tRNA selection and translation termination.

Biochemistry and Cell Biology-biochimie Et Biologie Cellulaire, 1995

Nature Structural Biology, 1997

Molecular Cell, 1999

protected by P site-bound tRNA (Moazed and Noller, Baltimore, Maryland 21205 1989). One of these ... more protected by P site-bound tRNA (Moazed and Noller, Baltimore, Maryland 21205 1989). One of these nucleotide protections led to the identification of a Watson-Crick base-pairing interaction between G2252 in domain V of 23S rRNA (a universally Summary conserved nucleotide) (Samaha et al., 1995) and C74 of P site bound tRNA. Interactions between other conserved The aminoacyl (A site) tRNA analog 4-thio-dT-p-C-pnucleotides of domain V and the universal A76 and C75 puromycin (s 4 TCPm) photochemically cross-links with of peptidyl tRNA remain elusive (Samaha et al., 1995; high efficiency and specificity to G2553 of 23S rRNA Spahn et al., 1996b; Green et al., 1997; Saarma et al., and is peptidyl transferase reactive in its cross-linked 1998) although a Hoogsteen pairing interaction between state, establishing proximity between the highly con-U2585 and A76 has been proposed (Porse et al., 1996). served 2555 loop in domain V of 23S rRNA and the In the A site, tRNA footprinting analysis similarly idenuniversally conserved CCA end of tRNA. To test for tified nucleotides in domain V of 23S rRNA that were base-pairing interactions between 23S rRNA and aminoprotected by A site-bound tRNA and that might thereacyl tRNA, site-directed mutations were made at the fore be involved in direct rRNA-tRNA interactions; these universally conserved nucleotides U2552 and G2553 positions included both G2553 and U2555 in the highly of 23S rRNA in both E. coli and B. stearothermophilus conserved 2555 loop (Moazed and Noller, 1989). Reribosomal RNA and incorporated into ribosomes. Mucently, we identified a highly specific and efficient crosstations at G2553 resulted in dominant growth defects in link between a photoactivated aminoacyl tRNA analog, E. coli and in decreased levels of peptidyl transferase 4-thio-dT-p-C-p-Puromycin (s 4 TCPm), and G2553 of activity in vitro. Genetic analysis in vitro of U2552 and 23S rRNA (Green et al., 1998); this cross-linked s 4 TCPm G2553 mutant ribosomes and CCA end mutant tRNA substrate is highly reactive in the peptidyl transferase substrates identified a base-pairing interaction bereaction. These data place the CCA end of A site tRNA tween C75 of aminoacyl tRNA and G2553 of 23S rRNA. in the vicinity of the 2555 loop of domain V of 23S rRNA (Figure 1). Two additional experiments place the 2555 loop near the CCA end of the A site tRNA: genetic studies * To whom correspondence should be addressed (e-mail: ragreen@ jhmi.edu). previously identified P site interaction (Samaha et al., Lieberman, K.R., and Dahlberg, A.E. (1995). Ribosome-catalyzed peptide-bond formation. Prog. Nucleic Acid Res. Mol. Biol. 50, 1-23. Thanks to H. Noller for the work initiated in his laboratory; to B. Merryman, C., Moazed, D., McWhirter, J., and Noller, H.F. (1999a). Cormack, K. Lieberman, and C. Greider for critical reading of the Nucleotides in 16S rRNA protected by the association of 30S and manuscript; to G. Culver for Figure 1B; to J. Puglisi, E. Blackburn, 50S ribosomal subunits. J. Mol. Biol. 285, 97-105. . rRNA in the ribosomal A, P, and E sites. Cell 57, 585-597. Monro, R.E., and Marcker, K.A. (1967). Ribosome-catalysed reaction of puromycin with a formylmethionine-containing oligonucleotide. References J. Mol. Biol. 25, 347-350. Newman, A.J., and Norman, C. (1992). U5 snRNA interacts with exon Ban, N., Nissen, P., Hansen, J., Capel, M., Moore, P.B., and Steitz, sequences at 5Ј and 3Ј splice sites. Cell 68, 743-754. T.A. (1999). Placement of protein and RNA structures into the 5 Å -resolution map of the 50S ribosomal subunit. Nature 400, 841-847. Noller, H.F., and Woese, C.R. (1981). Secondary structure of 16S ribosomal RNA. Science 212, 403-411. Bhuta, P., Kumar, G., and Chladek, S. (1982). The peptidyltransferase center of Escherichia coli ribosomes: binding sites for the cytidine Noller, H.F., Kop, J., Wheaton, V., Brosius, J., Gutell, R.R., Kopylov, 3Ј-phosphate residues of the aminoacyl-tRNA 3Ј-terminus and the A.M., Dohme, F., Herr, W., Stahl, D.A., Gupta, R., and Waese, C.R. interrelationships between the acceptor and donor sites. Biochim. (1981). Secondary structure model for 23S ribosomal RNA. Nucleic Acids Res. 9, 6167-6189. Biophys. Acta 696, 208-211.

Biochemistry, 1999

In vitro transcripts of Bacillus stearothermophilus 23S rRNA can be reconstituted into catalytica... more In vitro transcripts of Bacillus stearothermophilus 23S rRNA can be reconstituted into catalytically active 50S ribosomal subunits with an efficiency only 3-4-fold lower than that of natural 23S rRNA. Thus, post-transcriptional modifications in 23S rRNA are not essential for the assembly or function of the 50S subunit of the ribosome. This reconstitution sytem has been used to characterize the peptidyl transferase activity of site-directed mutations in 23S rRNA at positions G2252, U2506, U2584, and A2602 (Escherichia coli numbering), demonstrating its potential for the analysis of the role played by 23S rRNA in the function of the 50S subunit of the ribosome.

Nature, 1995

Interaction of the conserved CCA terminus of tRNA with rRNA in the peptidyl transferase P site ha... more Interaction of the conserved CCA terminus of tRNA with rRNA in the peptidyl transferase P site has been studied by in vitro genetics. A watson-Crick G-C pair between G2252 in a conserved hairpin loop of 23S rRNA and C74 at the acceptor end of tRNA is required for proper functional interaction of the CCA end of tRNA with the ribosomal P site. These findings establish a direct role for 23S rRNA in protein synthesis.

Proceedings of The National Academy of Sciences, 2001

On the basis of the recent atomic-resolution x-ray structure of the 50S ribosomal subunit, residu... more On the basis of the recent atomic-resolution x-ray structure of the 50S ribosomal subunit, residues A2451 and G2447 of 23S rRNA were proposed to participate directly in ribosome-catalyzed peptide bond formation. We have examined the peptidyltransferase and protein synthesis activities of ribosomes carrying mutations at these nucleotides. In Escherichia coli, pure mutant ribosome populations carrying either the G2447A or G2447C mutations maintained cell viability. In vitro, the G2447A ribosomes supported protein synthesis at a rate comparable to that of wild-type ribosomes. In single-turnover peptidyltransferase assays, G2447A ribosomes were shown to have essentially unimpaired peptidyltransferase activity at saturating substrate concentrations. All three base changes at the universally conserved A2451 conferred a dominant lethal phenotype when expressed in E. coli. Nonetheless, significant amounts of 2451 mutant ribosomes accumulated in polysomes, and all three 2451 mutations stimulated frameshifting and readthrough of stop codons in vivo. Furthermore, ribosomes carrying the A2451U transversion synthesized full-length ␤-lactamase chains in vitro. Pure mutant ribosome populations with changes at A2451 were generated by reconstituting Bacillus stearothermophilus 50S subunits from in vitro transcribed 23S rRNA. In single-turnover peptidyltransferase assays, the rate of peptide bond formation was diminished 3-to 14-fold by these mutations. Peptidyltransferase activity and in vitro ␤-lactamase synthesis by ribosomes with mutations at A2451 or G2447 were highly resistant to chloramphenicol. The significant levels of peptidyltransferase activity of ribosomes with mutations at A2451 and G2447 need to be reconciled with the roles proposed for these residues in catalysis.

Science, 1998

In the ribosome, the aminoacyl-transfer RNA (tRNA) analog 4-thio-dT-p-C-p-puromycin crosslinks ph... more In the ribosome, the aminoacyl-transfer RNA (tRNA) analog 4-thio-dT-p-C-p-puromycin crosslinks photochemically with G2553 of 23S ribosomal RNA (rRNA). This covalently linked substrate reacts with a peptidyl-tRNA analog to form a peptide bond in a peptidyl transferase-catalyzed reaction. This result places the conserved 2555 loop of 23S rRNA at the peptidyl transferase A site and suggests that peptide bond formation can occur uncoupled from movement of the A-site tRNA. Crosslink formation depends on occupancy of the P site by a tRNA carrying an intact CCA acceptor end, indicating that peptidyl-tRNA, directly or indirectly, helps to create the peptidyl transferase A site.

Proceedings of The National Academy of Sciences, 1999

Peptidyl transferase activity of Thermus aquaticus ribosomes is resistant to the removal of a sig... more Peptidyl transferase activity of Thermus aquaticus ribosomes is resistant to the removal of a significant number of ribosomal proteins by protease digestion, SDS, and phenol extraction. To define the upper limit for the number of macromolecular components required for peptidyl transferase, particles obtained by extraction of T. aquaticus large ribosomal subunits were isolated and their RNA and protein composition was characterized. Active subribosomal particles contained both 23S and 5S rRNA associated with notable amounts of eight ribosomal proteins. N-terminal sequencing of the proteins identified them as L2, L3, L13, L15, L17, L18, L21, and L22. Ribosomal protein L4, which previously was thought to be essential for the reconstitution of particles active in peptide bond formation, was not found. These findings, together with the results of previous reconstitution experiments, reduce the number of possible essential macromolecular components of the peptidyl transferase center to 23S rRNA and ribosomal proteins L2 and L3. Complete removal of ribosomal proteins from T. aquaticus rRNA resulted in loss of tertiary folding of the particles and inactivation of peptidyl transferase. The accessibility of proteins in active subribosomal particles to proteinase hydrolysis was increased significantly after RNase treatment. These results and the observation that 50S ribosomal subunits exhibited much higher resistance to SDS extraction than 30S subunits are compatible with a proposed structural organization of the 50S subunit involving an RNA ''cage'' surrounding a core of a subset of ribosomal proteins.

Annual Review of Biochemistry, 1997

The ribosome is a large multifunctional complex composed of both RNA and proteins. Biophysical me... more The ribosome is a large multifunctional complex composed of both RNA and proteins. Biophysical methods are yielding low-resolution structures of the overall architecture of ribosomes, and high-resolution structures of individual proteins and segments of rRNA. Accumulating evidence suggests that the ribosomal RNAs play central roles in the critical ribosomal functions of tRNA selection and binding, translocation, and peptidyl transferase. Biochemical and genetic approaches have identified specific functional interactions involving conserved nucleotides in 16S and 23S rRNA. The results obtained by these quite different approaches have begun to converge and promise to yield an unprecedented view of the mechanism of translation in the coming years.

Molecular Cell, 2003

Translocation of the mRNA:tRNA complex through the ribosome is promoted by elongation factor G (E... more Translocation of the mRNA:tRNA complex through the ribosome is promoted by elongation factor G (EF-G) during the translation cycle. Previous studies established that modification of ribosomal proteins with thiol-specific reagents promotes this event in the absence of EF-G. Here we identify two small subunit interface proteins S12 and S13 that are essential for maintenance of a pretranslocation state. Omission of these proteins using in vitro reconstitution procedures yields ribosomal particles that translate in the absence of enzymatic factors. Conversely, replacement of cysteine residues in these two proteins yields ribosomal particles that are refractive to stimulation with thiol-modifying reagents. These data support a model where S12 and S13 function as control elements for the more ancient rRNA- and tRNA-driven movements of translocation.

Journal of Molecular Biology, 2002

Translation of polyphenylalanine from a polyuridine template by the ribosome in the absence of th... more Translation of polyphenylalanine from a polyuridine template by the ribosome in the absence of the elongation factors EFG and EFTu (and the energy derived from GTP hydrolysis) is promoted by modification of the ribosome with thiol-specific reagents such as para-chloromercuribenzoate (pCMB). Here, we examine the translational cycle of modified ribosomes and show that peptide bond formation and tRNA binding are largely unaffected, whereas translocation of the mRNA:tRNA complex is substantially promoted by pCMB modification. The translocation movements that we observe are authentic by multiple criteria including the processivity of translation, accuracy of movement (three-nucleotide) along a defined mRNA template and sensitivity to antibiotics. Characterization of the modified ribosomes reveals that the protein content of the ribosomes is not depleted but that their subunit association properties are severely compromised. These data suggest that molecular targets (ribosomal proteins) in the interface region of the ribosome are critical barriers that influence the translocation of the mRNA:tRNA complex.

Molecular Cell, 2008

A crucial step in translation is the translocation of tRNAs through the ribosome. In the transiti... more A crucial step in translation is the translocation of tRNAs through the ribosome. In the transition from one canonical site to the other, the tRNAs acquire intermediate configurations, so-called hybrid states. At this stage, the small subunit is rotated with respect to the large subunit, and the anticodon stem loops reside in the A and P sites of the small subunit, while the acceptor ends interact with the P and E sites of the large subunit. In this work, by means of cryo-EM and particle classification procedures, we visualize the hybrid state of both A/P and P/E tRNAs in an authentic factor-free ribosome complex during translocation. In addition, we show how the repositioning of the tRNAs goes hand in hand with the change in the interplay between S13, L1 stalk, L5, H68, H69, and H38 that is caused by the ratcheting of the small subunit.

Cell, 2004

It is clear that a major contribution to catalysis of peptide bond formation by the ribosome deri... more It is clear that a major contribution to catalysis of peptide bond formation by the ribosome derives from simply positioning two reactive substrates in close proximity to one another in an orientation favorable for catalysis (Jencks, 1969). Direct base-pairing interactions be-Medicine tween the CCA ends of the tRNA substrates and rRNA Baltimore, Maryland 21205 elements, the A and P loops, clearly play a central role in positioning the substrates for catalysis (Kim and Green, 1999; Nissen et al., 2000; Samaha et al., 1995). Indeed, Summary it is possible that the major function of this well-organized and densely packed active site is to position reac-Peptide bond formation and peptide release are catative substrates and possibly "buttress" motion along the lyzed in the active site of the large subunit of the reaction coordinate (Rajagopalan and Benkovic, 2002). ribosome where universally conserved nucleotides However, other mechanisms for promoting this reaction surround the CCA ends of the peptidyl-and aminoacylmay also be utilized by the ribosome, including general tRNA substrates. Here, we describe the use of an afacid base, metal ion-assisted, substrate-assisted, or finity-tagging system for the purification of mutant electrostatic catalysis, as has been observed in other ribosomes and analysis of four universally conserved protein and RNA enzymes. nucleotides in the innermost layer of the active site: From a chemical perspective, peptide release is a A2451, U2506, U2585, and A2602. While pre-steadymore challenging reaction than peptide bond formation state kinetic analysis of the peptidyl transferase activbecause of the lower nucleophilicity of water relative to ity of the mutant ribosomes reveals substantially the primary amine of an amino acid. Our current underreduced rates of peptide bond formation using the standing of peptide release relies heavily on the obminimal substrate puromycin, their rates of peptide served conservation of a GGQ motif in class I release bond formation are unaffected when the substrates factors from eukaryotes to bacteria (Frolova et al., 1999). are intact aminoacyl-tRNAs. These mutant ribosomes Based on crystal structures of eRF1 and RF2 (Song et do, however, display substantial defects in peptide al., 2000; Vestergaard et al., 2001), it has been proposed release. These results reveal a view of the catalytic that this highly conserved motif plays a critical role in center in which an inner shell of conserved nucleotides coordinating a water molecule in the active site of the is pivotal for peptide release, while an outer shell is ribosome for participation in hydrolysis. Recent cryoEM responsible for promoting peptide bond formation. data and tethered chemical probing experiments have provided compelling evidence that this conserved GGQ

Molecular Cell, 2007

Peptide release on the ribosome is catalyzed in the large subunit peptidyl transferase center by ... more Peptide release on the ribosome is catalyzed in the large subunit peptidyl transferase center by release factors on recognition of stop codons in the small subunit decoding center. Here we examine the role of the decoding center in this process. Mutation of decoding center nucleotides or removal of 2 0 OH groups from the codon-deleterious in the related process of tRNA selection-has only mild effects on peptide release. The miscoding antibiotic paromomycin, which binds the decoding center and promotes the critical steps of tRNA selection, instead dramatically inhibits peptide release. Differences in the kinetic mechanism of paromomycin inhibition on stop and sense codons, paired with correlated structural changes monitored by chemical footprinting, suggest that recognition of stop codons by release factors induces specific structural rearrangements in the small subunit decoding center. We propose that, like other steps in translation, the specificity of peptide release is achieved through an induced-fit mechanism.