Andrey V. Zavialov, Vasili V. Hauryliuk, Måns Ehrenberg  Molecular Cell 

Slides:



Advertisements
Similar presentations
Translation – Initiation
Advertisements

Relationship between Genotype and Phenotype
Biochemical Specialization within Arabidopsis RNA Silencing Pathways
Fabien Darfeuille, Cecilia Unoson, Jörg Vogel, E. Gerhart H. Wagner 
R. Andrew Marshall, Colin Echeverría Aitken, Joseph D. Puglisi 
Kirill B. Gromadski, Marina V. Rodnina  Molecular Cell 
Reverse Translocation of tRNA in the Ribosome
Volume 33, Issue 2, Pages (January 2009)
Volume 28, Issue 3, Pages (November 2007)
Laura Lancaster, Harry F. Noller  Molecular Cell 
Relationship between Genotype and Phenotype
Smita Shankar, Asma Hatoum, Jeffrey W. Roberts  Molecular Cell 
A New Window onto Translational Repression by Bacterial sRNAs
Stop Codon Recognition by Release Factors Induces Structural Rearrangement of the Ribosomal Decoding Center that Is Productive for Peptide Release  Elaine.
Volume 46, Issue 4, Pages (May 2012)
Volume 37, Issue 1, Pages (January 2010)
Volume 51, Issue 2, Pages (July 2013)
The Role of ABCE1 in Eukaryotic Posttermination Ribosomal Recycling
Volume 18, Issue 6, Pages (June 2005)
Ben B. Hopkins, Tanya T. Paull  Cell 
Locking and Unlocking of Ribosomal Motions
Hani S. Zaher, Rachel Green  Molecular Cell 
Genetically Encoded but Nonpolypeptide Prolyl-tRNA Functions in the A Site for SecM- Mediated Ribosomal Stall  Hiroki Muto, Hitoshi Nakatogawa, Koreaki.
A Role for REP Sequences in Regulating Translation
Maintaining the Ribosomal Reading Frame
Volume 130, Issue 6, Pages (September 2007)
Shinobu Chiba, Koreaki Ito  Molecular Cell 
A Model for How Ribosomal Release Factors Induce Peptidyl-tRNA Cleavage in Termination of Protein Synthesis  Stefan Trobro, Johan Åqvist  Molecular Cell 
Volume 35, Issue 1, Pages (July 2009)
Trapping the Ribosome to Control Gene Expression
Hiro-oki Iwakawa, Yukihide Tomari  Molecular Cell 
Jingyi Fei, Pallav Kosuri, Daniel D. MacDougall, Ruben L. Gonzalez 
Different aa-tRNAs Are Selected Uniformly on the Ribosome
Fabien Darfeuille, Cecilia Unoson, Jörg Vogel, E. Gerhart H. Wagner 
More Than One Glycan Is Needed for ER Glucosidase II to Allow Entry of Glycoproteins into the Calnexin/Calreticulin Cycle  Paola Deprez, Matthias Gautschi,
Cap-Assisted Internal Initiation of Translation of Histone H4
Nature of the Nucleosomal Barrier to RNA Polymerase II
Volume 66, Issue 5, Pages e4 (June 2017)
Dissection of the Mechanism for the Stringent Factor RelA
Volume 129, Issue 5, Pages (June 2007)
Destabilization of the P Site Codon-Anticodon Helix Results from Movement of tRNA into the P/E Hybrid State within the Ribosome  Kevin G. McGarry, Sarah.
Volume 29, Issue 1, Pages (January 2008)
Sukhyun Kang, Megan D. Warner, Stephen P. Bell  Molecular Cell 
Cap-Assisted Internal Initiation of Translation of Histone H4
Volume 20, Issue 1, Pages (July 2017)
Volume 39, Issue 2, Pages (July 2010)
Caught on Camera: Intermediates of Ribosome Recycling
The Mammalian RNA Polymerase II C-Terminal Domain Interacts with RNA to Suppress Transcription-Coupled 3′ End Formation  Syuzo Kaneko, James L. Manley 
The Pathway of HCV IRES-Mediated Translation Initiation
Volume 58, Issue 5, Pages (June 2015)
Sichen Shao, Ramanujan S. Hegde  Molecular Cell 
Volume 112, Issue 1, Pages (January 2003)
DNA-Induced Switch from Independent to Sequential dTTP Hydrolysis in the Bacteriophage T7 DNA Helicase  Donald J. Crampton, Sourav Mukherjee, Charles.
tRNA Binds to Cytochrome c and Inhibits Caspase Activation
Volume 121, Issue 5, Pages (June 2005)
mRNA Helicase Activity of the Ribosome
Accurate Translocation of mRNA by the Ribosome Requires a Peptidyl Group or Its Analog on the tRNA Moving into the 30S P Site  Kurt Fredrick, Harry F.
Volume 9, Issue 1, Pages (January 2002)
Sequence of Steps in Ribosome Recycling as Defined by Kinetic Analysis
Regulation of mRNA Translation in Neurons—A Matter of Life and Death
How Initiation Factors Maximize the Accuracy of tRNA Selection in Initiation of Bacterial Protein Synthesis  Ayman Antoun, Michael Y. Pavlov, Martin Lovmar,
Volume 32, Issue 2, Pages (October 2008)
The Conformational Dynamics of the Mitochondrial Hsp70 Chaperone
A Minimal RNA Polymerase III Transcription System from Human Cells Reveals Positive and Negative Regulatory Roles for CK2  Ping Hu, Si Wu, Nouria Hernandez 
An SOS Inhibitor that Binds to Free RecA Protein: The PsiB Protein
A New Window onto Translational Repression by Bacterial sRNAs
Volume 9, Issue 1, Pages (January 2002)
Inhibition of Msh6 ATPase Activity by Mispaired DNA Induces a Msh2(ATP)-Msh6(ATP) State Capable of Hydrolysis-Independent Movement along DNA  Dan J. Mazur,
Volume 125, Issue 6, Pages (June 2006)
A RecA Filament Capping Mechanism for RecX Protein
Presentation transcript:

Splitting of the Posttermination Ribosome into Subunits by the Concerted Action of RRF and EF-G  Andrey V. Zavialov, Vasili V. Hauryliuk, Måns Ehrenberg  Molecular Cell  Volume 18, Issue 6, Pages 675-686 (June 2005) DOI: 10.1016/j.molcel.2005.05.016 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 P Site Bound tRNA Keeps the mRNA Reading Frame on the 30S Subunit (A–C) Three termination complexes with a tetra- (MFTI), penta- (MFTMI), and hexapeptide (MTMFTI) attached to the P site bound tRNAIle. (D–G) Schemes illustrating the initial and final positions of the three mRNAs in the postTC(MFTI), postTC(MFTMI), and postTC(MTMFTI) complexes as well as the fate of the mRNA SD-anti-SD contact in each case. In (G), tRNAfMet was added in trans, which changed the final position of the mRNA in relation to its final position in scheme (D). The schemes also illustrate how the mRNA positions were identified by A site-specific RelE cleavage. Molecular Cell 2005 18, 675-686DOI: (10.1016/j.molcel.2005.05.016) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 Stability of the Deacylated tRNA in the P Site Depends on the Distance from the SD Region on the mRNA (A) Time-dependent cleavage of mRNA in posttermination complexes. PostTC(MFTI) or postTC(MFTMI) was preincubated during varying times in the absence of RelE. Then, RelE was added to allow for mRNA cleavage during 15 s, and the cleavage products are shown in lane MFTI1 or MFTMI1, respectively. PostTC(MFTI) or postTC(MFTMI) was incubated during varying times in the presence of RelE, and the cleavage products are shown in lane MFTI2 or MFTMI2, respectively. In all cases, 0 time corresponds to RelE cuts of mRNA in pretermination ribosomal complexes, i.e., before peptide release by puromycin. AC*G cuts appear when tRNAIle has dissociated from the ribosome, allowing for mRNA movement in relation to the 30S subunit as explained in Figure 1D. (B) mRNA movement in the postTC(MFTMI) complex before and after addition of tRNAfMet in trans. The postTC(MFTMI) complex (220 nm) was incubated with RelE (80 mM) during varying times. At 3.5 min incubation time, tRNAfMet was added to a final concentration of 2 μM, and the samples were further incubated during varying times. Initially, the mRNA is cleaved in the stop codon (UA*G), then increasing cleavage in AC*G occurs during the first incubation period. After addition of tRNAfMet, cleavage occurs also in a UUU codon (U*UU and UU*U) of the mRNA. The results are further explained in Figure 1E (dissociation of tRNAIle) and Figure 1G (binding of tRNAfMet to the A site). (C) mRNA movement in the postTC(MFTI) complex in the presence of either tRNAfMet or fMet-tRNAfMet or in the absence of trans-added initiator tRNA. The postTC(MFTI) complex (210 nM) was incubated with RelE (80 nM) during 20 min in the absence of added tRNA (lane 1), in the presence of fMet-tRNAfMet (lane 2), or in the presence of tRNAfMet (lane 3). In the absence of any trans-added initiator tRNA, extensive cleavage only appears in the ACG to codon to AC*G. In the presence of either of the initiator tRNA variants, extensive double cleavage appears in the UUU codon. The data are further explained in Figures 1D and 1G. (D) The mRNA movement in the postTC(MTMFTI) complex in the absence or presence of tRNAfMet. The postTC(MTMFTI) complex (210 nM) was incubated with RelE (120 nM) during 15 min in the absence (lane c) or presence (lane d) of tRNAfMet. Lane a shows a control where the incubation was performed in the absence of RelE. Lane b shows another control, where the incubation was performed with a pretermination MTMFTI complex untreated by puromycin. These results are further explained in Figure 1F, illustrating why binding of tRNAfMet to the P site in this case does not shift the A site cleavage by RelE (compare with Figures 2C and 1G), simply because in this construct, the ACG codon (A site) is adjacent to the initiation AUG codon (P site). (E) Time dependence of the movement of the mRNA in the postTC(MTMFTI) complex. The postTC(MTMFTI) complex (200 nM) was incubated with RelE (120 nM) during varying incubation times. The figure illustrates the rapid movement of this mRNA relative to its reading frame, and the experiment is further explained in Figure 1F. (F) Time dependence of the release of [33P]tRNAIle from the P site of postTC(MFTI) and postTC(MTMFTI) ribosomes Molecular Cell 2005 18, 675-686DOI: (10.1016/j.molcel.2005.05.016) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 Probing of mRNA SD-Anti-SD Contacts during Translation of Short ORFs of Varying Lengths by trans-Acting Anti-SD DNA (A) Time-dependent cleavage of mRNA by RelE in the A site of postTC(MFTI) and postTC(MFTMI) complexes in the absence (MFTI1 or MFTMI1 lanes) or presence (MFTI2 or MFTMI2 lanes) of anti-SD DNA. The cleavage patterns do not change by the addition of anti-SD DNA, suggesting that the mRNA SD-anti-SD contacts remained intact during translation of the two ORFs. (B) Time-dependent cleavage of mRNA by RelE in the A site of the postTC(MTMFTI) complex in the absence (MTMFTI1 lanes) or presence (MTMFTI2 lanes) of anti-SD DNA. The RelE cleavage at the ACG codon was inhibited by addition of anti-SD DNA, suggesting that the mRNA SD-anti-SD contact was interrupted during translation of the ORF. (C) Time-dependent RelE cleavage of the postTC(MTMFTI) in the presence of tRNAfMet and in the absence (MTMFTI1 lanes) or presence (MTMFTI2 lanes) of anti-SD DNA. Molecular Cell 2005 18, 675-686DOI: (10.1016/j.molcel.2005.05.016) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 Dissociation of Deacylated tRNA from the P Site as Stimulated by EF-G and RRF (A) Time-dependent cleavage of mRNA by RelE in the A site of the postTC(MFTI) complex in the presence of varying combinations of translation factors. (B) Time-dependent release of [33P]tRNAIle from the P site of postTC(MFTI) in the presence of GTP and EF-G (●), RRF (▴), and RRF and EF-G (■). (C) Time-dependent release of [33P]tRNAIle from the P site of postTC(MFTI) in the presence of GTP, EF-G, and 2 μM IF3 (■), 5 μM RRF (▴), 5 μM RRF and 2 μM IF3 (■), 0.5 μM RRF (△), and 0.5 μM RRF and 2 μM IF3 (□). (D) Time-dependent release of tRNAIle from the P site of postTC(MFTI) detected by charging of tRNAIle with [14C]Ile by Ile-RS in the presence of EF-G, RRF, and GTP (●), GDPNP (▴), GDP (♦), and no nucleotides (■). The curves in Figure 3D describe the amount of tRNAIle released from the postTC as functions of time, whereas the curves in Figures 3B and 3C describe the fractions of tRNAIle remaining ribosome bound as functions of time. The amount of charged tRNAIle at time “t” in Figure 3D is denoted by “p(t).” The plateau value of charged Ile-tRNAIle at long incubation times is denoted by “p0.” If α(t) is defined as the fraction of tRNAIle remaining ribosome bound at “t,” as shown in Figures 3B and 3C, then p(t) and α(t) are connected by the simple relation: α(t) = 1 - p(t)/p0. Molecular Cell 2005 18, 675-686DOI: (10.1016/j.molcel.2005.05.016) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 Binding of EF-G•GDPNP and RRF to the 70S Ribosome and the 50S Subunit (A and B) Binding of EF-G•[3H]GDPNP to the 50S subunit monitored by nitrocellulose filtration in the presence of RRF (A) or in the presence of the 30S subunit (B). There was no detectable filter binding of GDPNP in the absence of EF-G and/or 50S subunits. (C) Binding of EF-G•[3H]GDPNP to the 50S subunit in the presence of RRF as a function of the [3H]GDPNP concentration. There was no detectable filter binding of GDPNP in the absence of EF-G or RRF or 50S subunits. (D) Binding of EF-G•[3H]GDPNP to the postTC(MFTI) ribosome in the presence of RRF. (E) Time-dependent binding of EF-G to the postTC(MFTI) in the presence or absence of RRF. (F) The rate of GTP hydrolysis on EF-G at varying concentrations of RRF in the presence of 50S subunits (▴) or in the presence of 50S and 30S subunits (●). Molecular Cell 2005 18, 675-686DOI: (10.1016/j.molcel.2005.05.016) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 6 EF-G•GTP and RRF Split the 70S Ribosome into Subunits (A–C) Effects of different factors on formation of ribosomal subunits. The reaction mixtures were applied to sucrose gradients, and ribosomes and subunits were separated from each other by ultracentrifuguation. In (A), 0.33 μM TC(MFTI) was incubated with 1.25 μM EF-G, 5 μM IF3, 0.3 mM GTP, 0.5 mM puromycin, and RRF at different concentrations. In (B), 0.33 μM TC(MFTI) was incubated with 1.25 μM EF-G, 8 μM RRF, 0.3 mM GTP, 0.5 mM puromycin, and IF3 at different concentrations. In (C), 0.33 μM TC(MFTI) was incubated with 1.25 EF-G, 12 μM RRF, 7 μM IF3, and 0.5 mM puromycin together with either one of the three nucleotides GTP, GDP, GDPNP (0.3 mM), or the buffer. (D) A scheme explaining the method of the experiment shown in (E). (E) Exchange of 30S subunits in 70S ribosomes in the presence of 1.25 μM EF-G, 5 μM RRF, and either one of the three nucleotides GTP, GDP, or GDPNP. Molecular Cell 2005 18, 675-686DOI: (10.1016/j.molcel.2005.05.016) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 7 A Model for Ribosome Splitting into Subunits by RRF, EF-G, and GTP (A) The postTC. (B) RRF binding to postTC induces rotation of the ribosomal subunits. In the resulting twisted state, the deacylated tRNA is bound in the hybrid P/E site making contact with protein L1. (C) EF-G•GDP binds to the ribosome in a twisted state, which catalyzes the GDP to GTP exchange on EF-G. (D) EF-G in the GTP form competes with RRF for binding to the A site of the ribosome. The head domain of RRF moves to the inside of the ribosome, disrupting the inter subunit bridges. (E and F) GTP hydrolysis on EF-G first results in the EF-G•GDP•Pi complex. After release of inorganic phosphate, EF-G adopts its GDP conformation, which leads to dissociation of the ribosomal subunits. (G) IF3 binds to the 30S subunit and prevents reformation of the 70S ribosome. (H) mRNA dissociates from the 30S subunit in complex with IF3. Molecular Cell 2005 18, 675-686DOI: (10.1016/j.molcel.2005.05.016) Copyright © 2005 Elsevier Inc. Terms and Conditions