P-site

The P-site (for peptidyl) is the second binding site for tRNA in the ribosome. The other two sites are the A-site (aminoacyl), which is the first binding site in the ribosome, and the E-site (exit), is the third and final binding site in the ribosome. During Protein translation, the P-site holds the tRNA which is linked to the growing polypeptide chain. When a stop codon is reached, the peptidyl-tRNA bond of the tRNA located in the P-site is cleaved releasing the newly synthesized protein.[1] During the translocation step of the elongation phase, the mRNA is advanced by one codon, coupled to movement of the tRNAs from the ribosomal A to P and P to E sites, catalyzed by elongation factor EF-G.[2]

Overview

The ribosomal P-site plays a vital role in all phases of translation. Initiation involves recognition of the start codon (AUG) by initiator tRNA in the P-site, elongation phase involves passage of many elongator tRNAs through the P site, termination phase involves hydrolysis of the mature polypeptide from tRNA bound to the P-site, and ribosome recycling involves release of deacylated tRNA. Binding a tRNA to the P-site in the presence of mRNA establishes codon-anticodon interaction and this interaction is important for small subunit ribosome (30S) contacts to the tRNA.[3]

The classical two-state model [4] proposes that ribosome contains two binding sites for tRNA, P-site and A-site. The A-site binds to incoming to aminoacyl-tRNA which has the anti-codon for the corresponding codon in the mRNA presented in the A-site. After peptide formation between the C-terminal carbonyl group of the growing polypeptide chain (attached to a P-site bound tRNA) and the amino group of the aminoacyl-tRNA (A-site bound), the polypeptide chain is then attached to the tRNA in the A-site. The deacylated tRNA remains in the P-site and gets released once the peptidyl-tRNA is transferred to the P-site.

Chemical modification experiments provided evidence of a hybrid model, where tRNAs can sample a hybrid state of binding during elongation phase (pre-translocation step). These hybrid states of binding are in which acceptor and anti-codon ends of tRNA are in different sites (A, P and E). Using Chemical probing methods, a set of phylogenetically conserved bases in ribosomal RNA where the tRNA binds has been examined and suggested to be directly involved in the binding of tRNA to the prokaryotic ribosome.[5] Correlation of such site specific protected bases in rRNA and occupancy of the A, P and E sites has allowed diagnostic assays of these bases to study the location of tRNA in any given state of the translational cycle. Authors proposed a hybrid model where higher affinity of the deactivated tRNA and peptide tRNA for the E and P sites of the 50S subunit, thermodynamically favour P/P to P/E and A/A to A/P transitions, which were further demonstrated through cryo-EM experiments.[6] Also, single molecule FRET studies have detected fluctuations in the positions of tRNAs,[7] leading to the conclusion that the classical (A/A-P/P) and hybrid states (A/P-P/E) of the tRNAs are certainly in dynamic equilibrium.

Prior to peptide bond formation, an aminoacyl-tRNA is bound in the A-site, a peptidyl-tRNA is bound in the P-site, and a deacylated tRNA (ready to exit from the ribosome) is bound to the E-site. Translation moves the tRNA from the A-site through the P- and E-sites, with the exception of the initiator tRNA, which binds directly to the P-site.[8] Recent experiments have reported that protein translation can also initiate from A-site. Using Toeprinting assay, it has been shown that Protein Synthesis initiates from the A-site of the Ribosome (Eukaryotic) in the cricket paralysis virus (CrPV). IGR-IRES (Intragenic regions-internal ribosome entry sites) can assemble 80S ribosomes from 40S and 60S ribosomal subunits in the absence of eIF2, Met-tRNAi, or GTP hydrolysis and without a coding triplet in the ribosomal P-site. Authors also showed IGR-IRES can direct translation of a protein whose N-terminal residue is not methionine.[9]

Structure

The complete three dimensional structure of the T. thermophilus 70S ribosome was determined using X-ray crystallography, containing mRNA and tRNAs bound to the P and E sites at 5.5 Å resolution and to the A site at 7 Å resolution. Authors found that all three tRNA binding sites (A, P, and E) of the ribosome contact all three respective tRNAs at universally conserved parts of their structures; this allows the ribosome to bind different tRNA species in precisely the same way. The translocation step of protein synthesis inescapably requires movements of 20 Å or more by the tRNAs, as they move from the A to P to E sites [10]

tRNA targeting antibiotics

Oxazolidines (e.g. linezolid) prevent the binding of the initiator tRNA at the P-site.[11] Oxazolidines have been demonstrated to pleiotropically affect initiator-tRNA binding, EF-P (elongation factor P) stimulated synthesis of peptide bonds, and EF-G-mediated translocation of initiator-tRNA into the P-site.[12]

Macrolide, Lincosamide and Streptogramin class of antibiotics prevent peptide bond formation and/or the translocation of tRNA from the A-site to the P-site on the ribosome [13][14] that eventually leads to interference with the elongation step and thus the inhibition of protein translation.

References

  1. ↑ Lodish, Harvey (2013). Molecular cell biology (Seventh ed.). New York: Worth Publ. pp. 141–143. ISBN 978-1429234139.
  2. ↑ Rodnina, MV; Savelsbergh, A; Katunin, VI; Wintermeyer, W (2 January 1997). "Hydrolysis of GTP by elongation factor G drives tRNA movement on the ribosome". Nature. 385 (6611): 37–41. doi:10.1038/385037a0. PMID 8985244.
  3. ↑ Schäfer, MA; Tastan, AO; Patzke, S; Blaha, G; Spahn, CM; Wilson, DN; Nierhaus, KH (24 May 2002). "Codon-anticodon interaction at the P site is a prerequisite for tRNA interaction with the small ribosomal subunit". The Journal of Biological Chemistry. 277 (21): 19095–105. doi:10.1074/jbc.M108902200. PMID 11867615.
  4. ↑ WATSON, JD (1964). "THE SYNTHESIS OF PROTEINS UPON RIBOSOMES". Bulletin de la Société de Chimie Biologique. 46: 1399–425. PMID 14270536.
  5. ↑ Moazed, D; Noller, HF (9 November 1989). "Intermediate states in the movement of transfer RNA in the ribosome". Nature. 342 (6246): 142–8. doi:10.1038/342142a0. PMID 2682263.
  6. ↑ Agirrezabala, Xabier; Lei, Jianlin; Brunelle, Julie L.; Ortiz-Meoz, Rodrigo F.; Green, Rachel; Frank, Joachim (October 2008). "Visualization of the Hybrid State of tRNA Binding Promoted by Spontaneous Ratcheting of the Ribosome". Molecular Cell. 32 (2): 190–197. doi:10.1016/j.molcel.2008.10.001.
  7. ↑ Blanchard, SC; Gonzalez, RL; Kim, HD; Chu, S; Puglisi, JD (October 2004). "tRNA selection and kinetic proofreading in translation". Nature Structural & Molecular Biology. 11 (10): 1008–14. doi:10.1038/nsmb831. PMID 15448679.
  8. ↑ Laursen, B. S.; Sorensen, H. P.; Mortensen, K. K.; Sperling-Petersen, H. U. (8 March 2005). "Initiation of Protein Synthesis in Bacteria". Microbiology and Molecular Biology Reviews. 69 (1): 101–123. doi:10.1128/MMBR.69.1.101-123.2005. PMC 1082788.
  9. ↑ Wilson, JE; Pestova, TV; Hellen, CU; Sarnow, P (18 August 2000). "Initiation of protein synthesis from the A site of the ribosome". Cell. 102 (4): 511–20. doi:10.1016/s0092-8674(00)00055-6. PMID 10966112.
  10. ↑ Yusupov, MM; Yusupova, GZ; Baucom, A; Lieberman, K; Earnest, TN; Cate, JH; Noller, HF (4 May 2001). "Crystal structure of the ribosome at 5.5 A resolution". Science. 292 (5518): 883–96. doi:10.1126/science.1060089. PMID 11283358.
  11. ↑ Chopra, Shaileja; Reader, John (25 December 2014). "tRNAs as Antibiotic Targets". International Journal of Molecular Sciences. 16 (1): 321–349. doi:10.3390/ijms16010321.
  12. ↑ Aoki, H.; Ke, L.; Poppe, S. M.; Poel, T. J.; Weaver, E. A.; Gadwood, R. C.; Thomas, R. C.; Shinabarger, D. L.; Ganoza, M. C. (1 April 2002). "Oxazolidinone Antibiotics Target the P Site on Escherichiacoli Ribosomes". Antimicrobial Agents and Chemotherapy. 46 (4): 1080–1085. doi:10.1128/AAC.46.4.1080-1085.2002. PMC 127084.
  13. ↑ Johnston, Nicole; Mukhtar, Tariq; Wright, Gerard (1 August 2002). "Streptogramin Antibiotics: Mode of Action and Resistance". Current Drug Targets. 3 (4): 335–344. doi:10.2174/1389450023347678.
  14. ↑ Champney, W. Scott; Tober, Craig L. (21 August 2000). "Specific Inhibition of 50S Ribosomal Subunit Formation in Staphylococcus aureus Cells by 16-Membered Macrolide, Lincosamide, and Streptogramin B Antibiotics". Current Microbiology. 41 (2): 126–135. doi:10.1007/s002840010106.
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