Aminoacyl-tRNA

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An aminoacyl-tRNA, with the tRNA above the arrow and a generic amino acid below the arrow. Most of the tRNA structure is shown as a simplified, colorful ball-and-stick model; the terminal adenosine and the amino acid are shown as structural formulas. The arrow indicates the ester linkage between the amino acid and tRNA. Aminoacyl-tRNA.png
An aminoacyl-tRNA, with the tRNA above the arrow and a generic amino acid below the arrow. Most of the tRNA structure is shown as a simplified, colorful ball-and-stick model; the terminal adenosine and the amino acid are shown as structural formulas. The arrow indicates the ester linkage between the amino acid and tRNA.

Aminoacyl-tRNA (also aa-tRNA or charged 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.

Contents

Alone, an amino acid is not the substrate necessary to allow for the formation of peptide bonds within a growing polypeptide chain. Instead, amino acids must be "charged" or aminoacylated with a tRNA to form their respective aa-tRNA. [1] Every amino acid has its own specific aminoacyl-tRNA synthetase, which is utilized to chemically bind to the tRNA that it is specific to, or in other words, "cognate" to. The pairing of a tRNA with its cognate amino acid is crucial, as it ensures that only the particular amino acid matching the anticodon of the tRNA, and in turn matching the codon of the mRNA, is used during protein synthesis.

In order to prevent translational errors, in which the wrong amino acid is incorporated into the polypeptide chain, evolution has provided for proofreading functionalities of aa-tRNA synthetases; these mechanisms ensure the proper pairing of an amino acid to its cognate tRNA. [2] Amino acids that are misacylated with the proper tRNA substrate undergo hydrolysis through the deacylation mechanisms possessed by aa-tRNA synthetases. [3]

Due to the degeneracy of the genetic code, multiple tRNAs will have the same amino acid but different anticodons. These different tRNAs are called isoacceptors. Under certain circumstances, non-cognate amino acids will be charged, resulting in mischarged or misaminoacylated tRNA. These mischarged tRNAs must be hydrolyzed in order to prevent incorrect protein synthesis.

While aa-tRNA serves primarily as the intermediate link between the mRNA coding strand and the encoded polypeptide chain during protein synthesis, it is also found that aa-tRNA have functions in several other biosynthetic pathways. aa-tRNAs are found to function as substrates in biosynthetic pathways for cell walls, antibiotics, lipids, and protein degradation.

It is understood that aa-tRNAs may function as donors of amino acids necessary for the modification of lipids and the biosynthesis of antibiotics. For example, microbial biosynthetic gene clusters may utilize aa-tRNAs in the synthesis of non-ribosomal peptides and other amino acid-containing metabolites. [4]

Synthesis

Aminoacyl-tRNA is produced in two steps. First, the adenylation of the amino acid, which forms aminoacyl-AMP:

Amino Acid + ATP → Aminoacyl-AMP + PPi

Second, the amino acid residue is transferred to the tRNA:

Aminoacyl-AMP + tRNA → Aminoacyl-tRNA + AMP

The overall net reaction is:

Amino Acid + ATP + tRNA → Aminoacyl-tRNA + AMP + PPi

The net reaction is energetically favorable only because the pyrophosphate (PPi) is later hydrolyzed. The hydrolysis of pyrophosphate to two molecules of inorganic phosphate (Pi) reaction is highly energetically favorable and drives the other two reactions. Together, these highly exergonic reactions take place inside the aminoacyl-tRNA synthetase specific for that amino acid. [5] [6]

Stability and hydrolysis

Research into the stability of aa-tRNAs illustrates that the acyl (or ester) linkage is the most important conferring factor, as opposed to the sequence of the tRNA itself. This linkage is an ester bond that chemically binds the carboxyl group of an amino acid to the terminal 3'-OH group of its cognate tRNA. [7] It has been discovered that the amino acid moiety of a given aa-tRNA provides for its structural integrity; the tRNA moiety dictates, for the most part, how and when the amino acid will be incorporated into a growing polypeptide chain. [8]

The different aa-tRNAs have varying pseudo-first-order rate constants for the hydrolysis of the ester bond between the amino acid and tRNA. [9] Such observations are due to, primarily, steric effects. Steric hindrance is provided for by specific side chain groups of amino acids, which aids in inhibiting intermolecular attacks on the ester carbonyl; these intermolecular attacks are responsible for hydrolyzing the ester bond.

Branched and aliphatic amino acids (valine and isoleucine) prove to generate the most stable aminoacyl-tRNAs upon their synthesis, with notably longer half lives than those that possess low hydrolytic stability (for example, proline). The steric hindrance of valine and isoleucine amino acids is generated by the methyl group on the β-carbon of the side chain. Overall, the chemical nature of the bound amino acid is responsible for determining the stability of the aa-tRNA. [10]

Increased ionic strength resulting from sodium, potassium, and magnesium salts has been shown to destabilize the aa-tRNA acyl bond. Increased pH also destabilizes the bond and changes the ionization of the α-carbon amino group of the amino acid. The charged amino group can destabilize the aa-tRNA bond via the inductive effect. [11] The elongation factor EF-Tu has been shown to stabilize the bond by preventing weak acyl linkages from being hydrolyzed. [12]

All together, the actual stability of the ester bond influences the susceptibility of the aa-tRNA to hydrolysis within the body at physiological pH and ion concentrations. It is thermodynamically favorable that the aminoacylation process yield a stable aa-tRNA molecule, thus providing for the acceleration and productivity of polypeptide synthesis. [13]

Drug targeting

Certain antibiotics, such as tetracyclines, prevent the aminoacyl-tRNA from binding to the ribosomal subunit in prokaryotes. It is understood that tetracyclines inhibit the attachment of aa-tRNA within the acceptor (A) site of prokaryotic ribosomes during translation. Tetracyclines are considered broad-spectrum antibiotic agents; these drugs exhibit capabilities of inhibiting the growth of both gram-positive and gram-negative bacteria, as well as other atypical microorganisms.

Furthermore, the TetM protein ( P21598 ) is found to allow aminoacyl-tRNA molecules to bind to the ribosomal acceptor site, despite being concentrated with tetracyclines that would typically inhibit such actions. The TetM protein is regarded as a ribosomal protection protein, exhibiting GTPase activity that is dependent upon ribosomes. Research has demonstrated that in the presence of TetM proteins, tetracyclines are released from ribosomes. Thus, this allows for aa-tRNA binding to the A site of ribosomes, as it is no longer precluded by tetracycline molecules. [14] TetO is 75% similar to TetM, and both have some 45% similarity with EF-G. The structure of TetM in complex with E. coli ribosome has been resolved. [15]

See also

Related Research Articles

<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.

<span class="mw-page-title-main">Aminoacyl tRNA synthetase</span> Class of enzymes

An aminoacyl-tRNA synthetase, also called tRNA-ligase, is an enzyme that attaches the appropriate amino acid onto its corresponding tRNA. It does so by catalyzing the transesterification of a specific cognate amino acid or its precursor to one of all its compatible cognate tRNAs to form an aminoacyl-tRNA. In humans, the 20 different types of aa-tRNA are made by the 20 different aminoacyl-tRNA synthetases, one for each amino acid of the genetic code.

Nonribosomal peptides (NRP) are a class of peptide secondary metabolites, usually produced by microorganisms like bacteria and fungi. Nonribosomal peptides are also found in higher organisms, such as nudibranchs, but are thought to be made by bacteria inside these organisms. While there exist a wide range of peptides that are not synthesized by ribosomes, the term nonribosomal peptide typically refers to a very specific set of these as discussed in this article.

In molecular biology, biosynthesis is a multi-step, enzyme-catalyzed process where substrates are converted into more complex products in living organisms. In biosynthesis, simple compounds are modified, converted into other compounds, or joined to form macromolecules. This process often consists of metabolic pathways. Some of these biosynthetic pathways are located within a single cellular organelle, while others involve enzymes that are located within multiple cellular organelles. Examples of these biosynthetic pathways include the production of lipid membrane components and nucleotides. Biosynthesis is usually synonymous with anabolism.

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.

Activation, in chemistry and biology, is the process whereby something is prepared or excited for a subsequent reaction.

<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.

Protein metabolism denotes the various biochemical processes responsible for the synthesis of proteins and amino acids (anabolism), and the breakdown of proteins by catabolism.

mRNA display

mRNA display is a display technique used for in vitro protein, and/or peptide evolution to create molecules that can bind to a desired target. The process results in translated peptides or proteins that are associated with their mRNA progenitor via a puromycin linkage. The complex then binds to an immobilized target in a selection step. The mRNA-protein fusions that bind well are then reverse transcribed to cDNA and their sequence amplified via a polymerase chain reaction. The result is a nucleotide sequence that encodes a peptide with high affinity for the molecule of interest.

<span class="mw-page-title-main">Eukaryotic translation termination factor 1</span> Protein-coding gene in the species Homo sapiens

Eukaryotic translation termination factor1 (eRF1), also referred to as TB3-1 or SUP45L1, is a protein that is encoded by the ERF1 gene. In Eukaryotes, eRF1 is an essential protein involved in stop codon recognition in translation, termination of translation, and nonsense mediated mRNA decay via the SURF complex.

<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">EF-G</span> Prokaryotic elongation factor

EF-G is a prokaryotic elongation factor involved in mRNA 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">Expanded genetic code</span> Modified genetic code

An expanded genetic code is an artificially modified genetic code in which one or more specific codons have been re-allocated to encode an amino acid that is not among the 22 common naturally-encoded proteinogenic amino acids.

<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.

Amino acid activation refers to the attachment of an amino acid to its respective transfer RNA (tRNA). The reaction occurs in the cell cytosol and consists of two steps: first, the enzyme aminoacyl tRNA synthetase catalyzes the binding of adenosine triphosphate (ATP) to a corresponding amino acid, forming a reactive aminoacyl adenylate intermediate and releasing inorganic pyrophosphate (PPi). Subsequently, aminoacyl tRNA synthetase binds the AMP-amino acid to a tRNA molecule, releasing AMP and attaching the amino acid to the tRNA. The resulting aminoacyl-tRNA is said to be charged.

The P-site 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. 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.

<span class="mw-page-title-main">Bottromycin</span> Chemical compound

Bottromycin is a macrocyclic peptide with antibiotic activity. It was first discovered in 1957 as a natural product isolated from Streptomyces bottropensis. It has been shown to inhibit methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE) among other Gram-positive bacteria and mycoplasma. Bottromycin is structurally distinct from both vancomycin, a glycopeptide antibiotic, and methicillin, a beta-lactam antibiotic.

References

  1. ↑ Peacock JR, Walvoord RR, Chang AY, Kozlowski MC, Gamper H, Hou YM (June 2014). "Amino acid-dependent stability of the acyl linkage in aminoacyl-tRNA". RNA. 20 (6): 758–64. doi:10.1261/rna.044123.113. PMC   4024630 . PMID   24751649.
  2. ↑ Kelly P, Ibba M (January 2018). "Aminoacyl-tRNA Quality Control Provides a Speedy Solution to Discriminate Right from Wrong". Journal of Molecular Biology. 430 (1): 17–19. doi: 10.1016/j.jmb.2017.10.025 . PMID   29111345.
  3. ↑ Francklyn CS, Mullen P (April 2019). "Progress and challenges in aminoacyl-tRNA synthetase-based therapeutics". The Journal of Biological Chemistry. 294 (14): 5365–5385. doi: 10.1074/jbc.REV118.002956 . PMC   6462538 . PMID   30670594.
  4. ↑ Ulrich EC, van der Donk WA (December 2016). "Cameo appearances of aminoacyl-tRNA in natural product biosynthesis". Current Opinion in Chemical Biology. 35: 29–36. doi:10.1016/j.cbpa.2016.08.018. PMC   5161580 . PMID   27599269.
  5. ↑ Swanson R, Hoben P, Sumner-Smith M, Uemura H, Watson L, Söll D (December 1988). "Accuracy of in vivo aminoacylation requires proper balance of tRNA and aminoacyl-tRNA synthetase". Science. 242 (4885): 1548–51. Bibcode:1988Sci...242.1548S. doi:10.1126/science.3144042. PMID   3144042.
  6. ↑ McClain WH (November 1993). "Rules that govern tRNA identity in protein synthesis". Journal of Molecular Biology. 234 (2): 257–80. doi:10.1006/jmbi.1993.1582. PMID   8230212.
  7. ↑ Kelly P, Ibba M (January 2018). "Aminoacyl-tRNA Quality Control Provides a Speedy Solution to Discriminate Right from Wrong". Journal of Molecular Biology. 430 (1): 17–19. doi: 10.1016/j.jmb.2017.10.025 . PMID   29111345.
  8. ↑ Francklyn CS, Mullen P (April 2019). "Progress and challenges in aminoacyl-tRNA synthetase-based therapeutics". The Journal of Biological Chemistry. 294 (14): 5365–5385. doi: 10.1074/jbc.REV118.002956 . PMC   6462538 . PMID   30670594.
  9. ↑ Hentzen D, Mandel P, Garel JP (October 1972). "Relation between aminoacyl-tRNA stability and the fixed amino acid". Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis. 281 (2): 228–32. doi:10.1016/0005-2787(72)90174-8. PMID   4629424.
  10. ↑ Kelly P, Ibba M (January 2018). "Aminoacyl-tRNA Quality Control Provides a Speedy Solution to Discriminate Right from Wrong". Journal of Molecular Biology. 430 (1): 17–19. doi: 10.1016/j.jmb.2017.10.025 . PMID   29111345.
  11. ↑ Schuber F, Pinck M (May 1974). "On the chemical reactivity of aminoacyl-tRNA ester bond. I. Influence of pH and nature of the acyl group on the rate of hydrolysis". Biochimie. 56 (3): 383–90. doi:10.1016/S0300-9084(74)80146-X. PMID   4853442.
  12. ↑ Peacock JR, Walvoord RR, Chang AY, Kozlowski MC, Gamper H, Hou YM (June 2014). "Amino acid-dependent stability of the acyl linkage in aminoacyl-tRNA". RNA. 20 (6): 758–64. doi:10.1261/rna.044123.113. PMC   4024630 . PMID   24751649.
  13. ↑ Peacock JR, Walvoord RR, Chang AY, Kozlowski MC, Gamper H, Hou YM (June 2014). "Amino acid-dependent stability of the acyl linkage in aminoacyl-tRNA". RNA. 20 (6): 758–64. doi:10.1261/rna.044123.113. PMC   4024630 . PMID   24751649.
  14. ↑ Chopra I, Roberts M (June 2001). "Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance". Microbiology and Molecular Biology Reviews. 65 (2): 232–60, second page, table of contents. doi:10.1128/MMBR.65.2.232-260.2001. PMC   99026 . PMID   11381101.
  15. ↑ Arenz, S; Nguyen, F; Beckmann, R; Wilson, DN (28 April 2015). "Cryo-EM structure of the tetracycline resistance protein TetM in complex with a translating ribosome at 3.9-Å resolution". Proceedings of the National Academy of Sciences of the United States of America. 112 (17): 5401–6. Bibcode:2015PNAS..112.5401A. doi: 10.1073/pnas.1501775112 . PMC   4418892 . PMID   25870267.
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