P-site

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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), the third. 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]

Contents

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 involves passage of many elongator tRNAs through the P site, termination 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 the ribosome contains two binding sites for tRNA, P-site and A-site. The A-site binds to incoming 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 is released once the peptidyl-tRNA is transferred to the P-site. How is the translocation of the peptidyl-tRNA from the A-site to the P-site achieved to complete the cycle? It was proposed that this is done in two steps by the movement of the two ribosomal subunits with respect to each other, with the formation of an intermediate hybrid structure: the A-site of one subunit with the P-site of the other subunit. [5] This is analogous to moving a large object: you move one end first, then the other.

Chemical modification experiments provided evidence of this hybrid model, in which tRNAs can sample a hybrid state of binding during the elongation phase (pre-translocation step). In these hybrid states of binding, 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 is suggested to be directly involved in the binding of tRNA to the prokaryotic ribosome. [6] 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 in which higher affinity of the deactivated tRNA and peptide tRNA for the E and P sites of the 50S subunit, thermodynamically favours P/P to P/E and A/A to A/P transitions, which were further demonstrated through cryo-EM experiments. [7] Also, single molecule FRET studies have detected fluctuations in the positions of tRNAs, [8] 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. [9] Recent experiments have reported that protein translation can also initiate from the 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. [10]

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 requires movements of 20 Å or more by the tRNAs, as they move from the A to P to E sites [11]

tRNA-targeting antibiotics

Oxazolidines (e.g. linezolid) prevent the binding of the initiator tRNA at the P-site. [12] 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. [13]

Macrolide, lincosamide and streptogramin classes of antibiotics prevent peptide bond formation and/or the translocation of tRNA from the A-site to the P-site on the ribosome [14] [15] that eventually leads to interference with the elongation step and thus the inhibition of protein translation.

Related Research Articles

<span class="mw-page-title-main">Ribosome</span> Intracellular organelle consisting of RNA and protein functioning to synthesize proteins

Ribosomes are macromolecular machines, found within all cells, that perform biological protein synthesis. Ribosomes link amino acids together in the order specified by the codons of messenger RNA (mRNA) molecules to form polypeptide chains. Ribosomes consist of two major components: the small and large ribosomal subunits. Each subunit consists of one or more ribosomal RNA (rRNA) molecules and many ribosomal proteins. The ribosomes and associated molecules are also known as the translational apparatus.

<span class="mw-page-title-main">Translation (biology)</span> Cellular process of protein synthesis

In biology, translation is the process in living cells in which proteins are produced using RNA molecules as templates. The generated protein is a sequence of amino acids. This sequence is determined by the sequence of nucleotides in the RNA. The nucleotides are considered three at a time. Each such triple results in addition of one specific amino acid to the protein being generated. The matching from nucleotide triple to amino acid is called the genetic code. The translation is performed by a large complex of functional RNA and proteins called ribosomes. The entire process is called gene expression.

<span class="mw-page-title-main">Transfer RNA</span> RNA that facilitates the addition of amino acids to a new protein

Transfer RNA is an adaptor molecule composed of RNA, typically 76 to 90 nucleotides in length, that serves as the physical link between the mRNA and the amino acid sequence of proteins. Transfer RNA (tRNA) does this by carrying an amino acid to the protein synthesizing machinery of a cell called the ribosome. Complementation of a 3-nucleotide codon in a messenger RNA (mRNA) by a 3-nucleotide anticodon of the tRNA results in protein synthesis based on the mRNA code. As such, tRNAs are a necessary component of translation, the biological synthesis of new proteins in accordance with the genetic code.

The peptidyl transferase is an aminoacyltransferase as well as the primary enzymatic function of the ribosome, which forms peptide bonds between adjacent amino acids using tRNAs during the translation process of protein biosynthesis. The substrates for the peptidyl transferase reaction are two tRNA molecules, one bearing the growing peptide chain and the other bearing the amino acid that will be added to the chain. The peptidyl chain and the amino acids are attached to their respective tRNAs via ester bonds to the O atom at the CCA-3' ends of these tRNAs. Peptidyl transferase is an enzyme that catalyzes the addition of an amino acid residue in order to grow the polypeptide chain in protein synthesis. It is located in the large ribosomal subunit, where it catalyzes the peptide bond formation. It is composed entirely of RNA. The alignment between the CCA ends of the ribosome-bound peptidyl tRNA and aminoacyl tRNA in the peptidyl transferase center contribute to its ability to catalyze these reactions. This reaction occurs via nucleophilic displacement. The amino group of the aminoacyl tRNA attacks the terminal carboxyl group of the peptidyl tRNA. Peptidyl transferase activity is carried out by the ribosome. Peptidyl transferase activity is not mediated by any ribosomal proteins but by ribosomal RNA (rRNA), a ribozyme. Ribozymes are the only enzymes which are not made up of proteins, but ribonucleotides. All other enzymes are made up of proteins. This RNA relic is the most significant piece of evidence supporting the RNA World hypothesis.

Bacterial translation is the process by which messenger RNA is translated into proteins in bacteria.

Eukaryotic translation is the biological process by which messenger RNA is translated into proteins in eukaryotes. It consists of four phases: initiation, elongation, termination, and recapping.

<span class="mw-page-title-main">Aminoacyl-tRNA</span>

Aminoacyl-tRNA is tRNA to which its cognate amino acid is chemically bonded (charged). The aa-tRNA, along with particular elongation factors, deliver the amino acid to the ribosome for incorporation into the polypeptide chain that is being produced during translation.

<span class="mw-page-title-main">EF-Tu</span> Prokaryotic elongation factor

EF-Tu is a prokaryotic elongation factor responsible for catalyzing the binding of an aminoacyl-tRNA (aa-tRNA) to the ribosome. It is a G-protein, and facilitates the selection and binding of an aa-tRNA to the A-site of the ribosome. As a reflection of its crucial role in translation, EF-Tu is one of the most abundant and highly conserved proteins in prokaryotes. It is found in eukaryotic mitochondria as TUFM.

A bacterial initiation factor (IF) is a protein that stabilizes the initiation complex for polypeptide translation.

<span class="mw-page-title-main">Prokaryotic large ribosomal subunit</span>

50S is the larger subunit of the 70S ribosome of prokaryotes, i.e. bacteria and archaea. It is the site of inhibition for antibiotics such as macrolides, chloramphenicol, clindamycin, and the pleuromutilins. It includes the 5S ribosomal RNA and 23S ribosomal RNA.

<span class="mw-page-title-main">Prokaryotic small ribosomal subunit</span> Smaller subunit of the 70S ribosome found in prokaryote cells

The prokaryotic small ribosomal subunit, or 30S subunit, is the smaller subunit of the 70S ribosome found in prokaryotes. It is a complex of the 16S ribosomal RNA (rRNA) and 19 proteins. This complex is implicated in the binding of transfer RNA to messenger RNA (mRNA). The small subunit is responsible for the binding and the reading of the mRNA during translation. The small subunit, both the rRNA and its proteins, complexes with the large 50S subunit to form the 70S prokaryotic ribosome in prokaryotic cells. This 70S ribosome is then used to translate mRNA into proteins.

<span class="mw-page-title-main">23S ribosomal RNA</span> A component of the large subunit of the prokaryotic ribosome

The 23S rRNA is a 2,904 nucleotide long component of the large subunit (50S) of the bacterial/archean ribosome and makes up the peptidyl transferase center (PTC). The 23S rRNA is divided into six secondary structural domains titled I-VI, with the corresponding 5S rRNA being considered domain VII. The ribosomal peptidyl transferase activity resides in domain V of this rRNA, which is also the most common binding site for antibiotics that inhibit translation, making it a target for ribosomal engineering. A well-known member of this antibiotic class, chloramphenicol, acts by inhibiting peptide bond formation, with recent 3D-structural studies showing two different binding sites depending on the species of ribosome. Numerous mutations in domains of the 23S rRNA with Peptidyl transferase activity have resulted in antibiotic resistance. 23S rRNA genes typically have higher sequence variations, including insertions and/or deletions, compared to other rRNAs.

<span class="mw-page-title-main">EF-G</span> Prokaryotic elongation factor

EF-G is a prokaryotic elongation factor involved in protein translation. As a GTPase, EF-G catalyzes the movement (translocation) of transfer RNA (tRNA) and messenger RNA (mRNA) through the ribosome.

<span class="mw-page-title-main">Protein synthesis inhibitor</span> Inhibitors of translation

A protein synthesis inhibitor is a compound that stops or slows the growth or proliferation of cells by disrupting the processes that lead directly to the generation of new proteins.

Streptogramin A is a group of antibiotics within the larger family of antibiotics known as streptogramins. They are synthesized by the bacteria Streptomyces virginiae. The streptogramin family of antibiotics consists of two distinct groups: group A antibiotics contain a 23-membered unsaturated ring with lactone and peptide bonds while group B antibiotics are depsipeptides. While structurally different, these two groups of antibiotics act synergistically, providing greater antibiotic activity than the combined activity of the separate components. These antibiotics have until recently been commercially manufactured as feed additives in agriculture, although today there is increased interest in their ability to combat antibiotic-resistant bacteria, particularly vancomycin-resistant bacteria.

Streptogramin B is a subgroup of the streptogramin antibiotics family. These natural products are cyclic hexa- or hepta depsipeptides produced by various members of the genus of bacteria Streptomyces. Many of the members of the streptogramins reported in the literature have the same structure and different names; for example, pristinamycin IA = vernamycin Bα = mikamycin B = osteogrycin B.

Translational regulation refers to the control of the levels of protein synthesized from its mRNA. This regulation is vastly important to the cellular response to stressors, growth cues, and differentiation. In comparison to transcriptional regulation, it results in much more immediate cellular adjustment through direct regulation of protein concentration. The corresponding mechanisms are primarily targeted on the control of ribosome recruitment on the initiation codon, but can also involve modulation of peptide elongation, termination of protein synthesis, or ribosome biogenesis. While these general concepts are widely conserved, some of the finer details in this sort of regulation have been proven to differ between prokaryotic and eukaryotic organisms.

<span class="mw-page-title-main">Elongation factor P</span>

EF-P is an essential protein that in bacteria stimulates the formation of the first peptide bonds in protein synthesis. Studies show that EF-P prevents ribosomes from stalling during the synthesis of proteins containing consecutive prolines. EF-P binds to a site located between the binding site for the peptidyl tRNA and the exiting tRNA. It spans both ribosomal subunits with its amino-terminal domain positioned adjacent to the aminoacyl acceptor stem and its carboxyl-terminal domain positioned next to the anticodon stem-loop of the P site-bound initiator tRNA. The EF-P protein shape and size is very similar to a tRNA and interacts with the ribosome via the exit “E” site on the 30S subunit and the peptidyl-transferase center (PTC) of the 50S subunit. EF-P is a translation aspect of an unknown function, therefore It probably functions indirectly by altering the affinity of the ribosome for aminoacyl-tRNA, thus increasing their reactivity as acceptors for peptidyl transferase.

In molecular biology, VAR1 protein domain, otherwise known as variant protein 1, is a ribosomal protein that forms part of the small ribosomal subunit in yeast mitochondria. Mitochondria possess their own ribosomes responsible for the synthesis of a small number of proteins encoded by the mitochondrial genome. VAR1 is the only protein in the yeast mitochondrial ribosome to be encoded in the mitochondria - the remaining approximately 80 ribosomal proteins are encoded in the nucleus. VAR1 along with 15S rRNA are necessary for the formation of mature 37S subunits.

Ribosomal L28e protein family is a family of evolutionarily related proteins. Members include 60S ribosomal protein L28.

References

  1. ↑ Lodish, Harvey (2013). Molecular cell biology (Seventh ed.). New York: Worth Publ. pp. 141–143. ISBN   978-1-4292-3413-9.
  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): 19095–19105. 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: 1399–1425. PMID   14270536.
  5. ↑ Bretscher, MS (1968). "Translocation in protein synthesis: a hybrid structure model". Nature. 218 (5142): 675–677. doi:10.1038/218675a0. PMID   5655957.
  6. ↑ Moazed, D; Noller, HF (9 November 1989). "Intermediate states in the movement of transfer RNA in the ribosome". Nature. 342 (6246): 142–148. doi:10.1038/342142a0. PMID   2682263.
  7. ↑ 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. PMC   2614368 . PMID   18951087.
  8. ↑ 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): 1008–1014. doi:10.1038/nsmb831. PMID   15448679.
  9. ↑ 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 . PMID   15755955.
  10. ↑ 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): 511–520. doi: 10.1016/s0092-8674(00)00055-6 . PMID   10966112.
  11. ↑ 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): 883–896. doi: 10.1126/science.1060089 . PMID   11283358.
  12. ↑ Chopra, Shaileja; Reader, John (25 December 2014). "tRNAs as Antibiotic Targets". International Journal of Molecular Sciences. 16 (1): 321–349. doi: 10.3390/ijms16010321 . PMC   4307249 . PMID   25547494.
  13. ↑ 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 . PMID   11897593.
  14. ↑ 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.
  15. ↑ 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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