Translation and protein synthesis

Gene expression is the process by which genetic information flows from DNA to RNA to the protein. The translation of DNA into RNA is termed transcription; protein synthesis from RNA templates is called translation. Details on gene expression and transcription can be found in a separate article.

Translation is carried out by ribosomes, which are large molecular complexes of ribosomal RNA (rRNA) and proteins. Ribosomes bind to RNA templates, also termed messenger RNA (mRNA), and catalyze the formation of a polypeptide based on this template. In the process, a charged transfer RNA (tRNA) recognizes a nucleotide triplet of mRNA that matches a specific amino acid (AA). The new AA is then linked to the next AA of the growing polypeptide on the ribosome. Translation ends once a specific nucleotide sequence of the mRNA is reached (a stop codon). The ribosome subsequently dissociates and the mRNA and newly synthesized protein are released. Before proteins are functional, a proper shape and destination are both necessary. Proteins begin to fold into their three-dimensional structure during translation according to the AA sequence and local chemical forces and reactions. Various specialized proteins (folding catalysts, chaperones) also help the newly formed proteins to fold properly and reach their correct destinations (e.g., cytosol, organelles, extracellular matrix) via protein modifications. The translation rate of proteins is adjusted to the current conditions of the cell and bodily demands, and is affected by the presence or absence of certain nutrients.

The genetic code

• Description: relationship between the DNA (or mRNA) nucleotide sequence and the respective amino acid sequence of a protein
• Codons
  • Codon: sequence of three nucleotides (a triplet) of mRNA that codes for a specific AA
  • Anticodon: sequence of three nucleotides in tRNA that is complementary to the codon on mRNA
  • The codon and anticodon are always paired in an antiparallel manner.
  • There are 64 combinations of codons: 61 for 20 AAs (including the start codon), and 3 for stop codons
  • Notable codons
    • Start codon: AUG (located at the 5' end of mRNA), initiates translation of the mRNA
      • Eukaryotes: code for methionine-tRNA (each polypeptide initially starts with methionine, which usually gets removed during translation)
      • Prokaryotes: code for N-formylmethionine (fMet; stimulates chemotaxis of neutrophils).
    • Stop codons: UGA, UAG, or UAA end translation

Stop codons (UAA, UGA, UAG): U Are Away, U Go Away, and U Are Gone.

Start codon: AUG AUthorizes Growth.

• Description: binding of tRNA to its corresponding AA
• Relevant enzymes: aminoacyl-tRNA synthetases
  • A group of tRNA-specific enzymes that catalyze tRNA charging
  • There is at least one synthetase for each proteinogenic AA.
• Reaction mechanism
  1. AA + ATP ; → aminoacyl-AMP + PPi
  2. tRNA + aminoacyl-AMP → aminoacyl-tRNA + AMP
• Mischarged tRNA
  • AAs are checked before and after binding to tRNA by the aminoacyl-tRNA synthetase. If an incorrect AA binds the tRNA, the bond is hydrolyzed.
  • Any mischarged tRNA inserts the wrong AA into the polypeptide, which is why AA-tRNA synthetase is important for accurate AA selection

Translation occurs in three phases in a functional ribosome: initiation, elongation, and termination; . It requires mRNA, tRNA, and rRNA.

Ribosome binding sites

• See “Ribosomes” for more details.
• The ribosome is composed of rRNA and proteins that form subunits.
  • Small subunit: 40S subunit in eukaryotes, 30S subunit in prokaryotes
  • Large subunit: 60S subunit in eukaryotes, 50S subunit in prokaryotes
• Binding site for mRNA is on the small ribosomal subunit.
• Both ribosomal subunits jointly form the binding sites for tRNA.
  • Aminoacyl site (A site)
  • Peptidyl site (P site)
  • Exit site (E site)
• Because of the length of most mRNA, more than one ribosome can bind them, allowing synthesis of multiple polypeptides at once = polysome.

Eukaryotes have Even-numbered ribosomal subunits (40S + 60S → 80S).
PrOkaryotes have Odd-numbered ribosomal subunits (30S + 50S → 70S).

For binding sites, think of a Growing APE party:
• Growing = GTP as the energy source
• A site: Arrival with Aminoacyl-tRNA
• P site: party of Peptides
• E site: party Ends and is Empty;tRNA Exits

Initiation

• Description: assembly of functional ribosomes with the help of initiation factors (IFs) and recognition of the start codon (AUG) on the mature mRNA by the initiator methionyl-tRNA (met-tRNA)
• Process
  1. Initiator met-tRNA, eukaryotic IF2 (eIF2), and GTP bind to the small ribosomal subunit to form a preinitiation complex (initially a 43s preinitiation complex).
     • eIF2: a small G protein
       • Binds initiator met-tRNA (ternary complex) and forms the final initiation complex by hydrolyzing GTP to GDP
       • Reconverted to the GTP-bound form by the guanine nucleotide exchange factor eIF2B
  2. mRNA is recognized by eIF4 and binds to the preinitiation complex, leading to the formation of the 48s preinitiation complex; eIF4 recognizes mRNA
     • Usually at the 5' cap in eukaryotes
     • Sometimes at an internal ribosome entry site (IRES)
       • A site of mRNA that allows translation initiation without a 5' cap
       • Most commonly located in the 5' UTR (especially of RNA viruses like poliovirus), but can be located at many sites in the mRNA and also occurs in eukaryotes
     • Initiator met-tRNA recognizes the start codon (typically the first AUG triplet after the 5' cap of the mRNA) and binds the P site.
     • Shine-Dalgarno sequence: ribosomal binding site contained in prokaryotic mRNA that enables the initiation of protein synthesis by aligning start codon and ribosome
       • Generally found ∼ 8 bases upstream of start codon
       • Common in bacteria
  3. GTP hydrolysis provides energy for the release of eIF2, allowing the large and small ribosomal subunits to assemble into a functional ribosome (the final initiation complex; 80S in eukaryotes, 70S in prokaryotes).

Elongation

• Directions of processes
  • mRNA is read in a 5' to 3' direction
  • Elongation occurs in an N-terminus to C-terminus direction: The N-terminus of the growing protein also initially leaves the ribosome.
• Process
  • Initiator met-tRNA is located at the P-site, or another previously matching aminoacyl-tRNA is bound there.
  • An aminoacyl-tRNA complex with eukaryotic elongation factor 1 (eEF1) hydrolyzes GTP, thereby releasing eEF1 and GDP and providing the energy for aminoacyl-tRNA to bind the A site (anticodon matches the codon of the mRNA).
  • The polypeptide is elongated by the stepwise addition of AAs via peptide bonds between the AAs bound to the A-sites and P-site (via tRNA).
  • Bond created by a dehydration reaction catalyzed by a peptidyl transferase that is intrinsic to the rRNA (“ribozyme”) of the large complex
  • Ribosomal translocation
    • The ribosome moves one triplet along the mRNA in the 3' direction.
      • Energy is derived from GTP hydrolysis that is catalyzed by eEF2.
    • After translocation, the tRNA that was in the A-site is now in the P-site, and the tRNA that was in the P-site is now in the E-site.
      • The A site is free again and can bind to a new aminoacyl-tRNA (in a complex with eEF1/GTP).
      • A peptidyl-tRNA is present at the P site with the growing peptide chain.
      • The unloaded tRNA is located at the E site.
    • The unloaded tRNA is released from the E-site.

Termination

• A release factor recognizes the stop codon, halts translation, and hydrolytically cleaves the peptidyl tRNA bonds (requires GTP), leading to release of the protein.

ATP for Activating (charging) tRNA and GTP for tRNA Gripping and Going through the ribosome (translocation) for Growing a polypeptide.

Protein folding

• Description: The process by which a protein goes from an unfolded native state to form a three-dimensional structure via progressive stabilization of the intermediate states until the most favorable energy level is achieved.
  • Correct protein folding begins during translation and is required for a protein to perform its function within the cell or organism.
• Intrinsic factors
  • The spatial structure is specified in the AA sequence of the protein (see protein structure for more details).
  • Largely driven by hydrophobic interactions, Van der Waals forces, H⁺-bonds, salt bridges, disulfide bonds
• Regulatory proteins: proteins that help other proteins to form their native structure
  • Chaperone proteins
    • Intracellular protein complexes that prevent protein aggregation during synthesis (thus prevent making proteins nonfunctional) and permit refolding of misfolded proteins in a protected environment
    • Assist in transporting (precursor) proteins
    • ATP is consumed in the process.
    • Example: heat shock proteins (e.g., Hsp70, Hsp60, Hsp90)
      • Constitutively expressed proteins that prevent denaturation or misfolding
      • Conditions that lead to increased expression include high temperatures, chemical stress, and hypoxia
  • Folding catalysts: enzymes that accelerate rate-limiting steps during protein folding
    • Protein disulfide-isomerase: Catalyzes the formation of thermodynamically favorable disulfide bonds within proteins (in proteins that possess more than two cysteine residues), if less energetically favorable disulfide bonds are formed.
    • Prolyl isomerase: Assists in finding the energetically favorable conformation of a peptide bond with the AA proline.

Protein misfolding

• Description: lack of folding or non-native protein folding as a result of denaturing factors
• Triggers
  • Stress: e.g., oxidative conditions
  • Increased body temperature (fever)
  • Mutations: e.g., chloride channel misfolding in cystic fibrosis; misfolded hemoglobin in sickle cell anemia; amyloidosis
• Mechanisms of misfolding
  • Hydrophobic amino acid residues on the surface of a misfolded protein tend to aggregate with other hydrophobic surfaces/proteins.
  • Accumulation of proteins rich in β-sheets can lead to fibrillation.
• Intracellular reaction: Misfolded proteins are identified and either rescued by chaperone proteins or are ubiquitinated and labeled for proteasomal degradation (see “Protein degradation”).
• Consequences of misfolding
  • In some disorders, the protective mechanism fails.
  • Misfolded proteins form insoluble aggregates and fibrils, which leads to cell damage and death (e.g., Parkinson disease and Alzheimer disease).
  • See “Protein degradation.”

Overview

• Many proteins require specific covalent alterations (co- or posttranslational modifcations) in addition to correct folding to function properly.
• Examples include glycosylation, lipid anchors, phosphorylation, acetylation, ubiquitination, ADP-ribosylation, biotinylation, carboxylation, methylation, and hydroxylation.

Protein glycosylation

• Enzymatic glycosylation involves the attachment of a carbohydrate to specific AA side chains of proteins via N-glycosidic (N-linked glycosylation) or O-glycosidic bonds (O-linked glycosylation), forming a glycoprotein
• Glycoproteins are usually cell membrane, lysosomal, or secretory proteins (e.g., serum proteins such as erythropoietin).

Sugar attachment to the asparagine residue of proteins (i.e., N-linked glycosylation) begins in the rough ER.

Enzymatic glycosylation should not be confused with nonenzymatic glycation. In glycation, aldoses (e.g., glucose) spontaneously bind to the amino groups of proteins and may influence their function. A classic example is HbA1c, whose function is unaffected by glycation.

Lipid anchors

• Description
  • Most membrane proteins interact with lipid membranes via hydrophobic side chains (e.g., valine or leucine residues)
  • However, lipid-anchored proteins are modified to covalently bind lipid anchors.
• Types
  • Acylation: linkage with long chain fatty acids, e.g., palmitic acid
  • Isoprenylation: linkage of a cysteine side chain of the protein with polyisoprene via a thioester bond, such as in:
    • Farnesylation: linkage with a farnesyl residue (three isoprene units, total of 15 C atoms)
    • Geranylgeranylation: linkage with a geranylgeranyl residue (four isoprene units, total of 20 C atoms)
  • GPI anchor: linkage with glycosylphosphatidylinositol (GPI), a glycolipid

Reversible covalent alterations

Enzymatic reversible protein modification alters the protein's spatial structure (conformation), thereby allowing its activity to be regulated. For example, a protein may interact with other proteins and/or become recognizable as a substrate. Reversible protein modification essentially allows the protein to be switched on or off.

• Phosphorylation: attachment of phosphate residues to the hydroxyl group (OH group) of serine or threonine or of tyrosine residues by kinases
  • Example: regulation of glycogen phosphorylase in glycogen metabolism via phosphorylation
  • Opposite: dephosphorylation regulated by phosphatases
• Acetylation: linkage with a CO-CH₃ group by acetyltransferases
  • Example: regulation of DNA condensation and with it transcription via acetylation of histone proteins
  • Opposite: deacetylation
• Ubiquitination: attachment of ubiquitin (a small protein) to the ε-amino group of lysine residues in proteins, especially in proteins to be degraded (e.g., cell cycle regulation via ubiquitination and degradation of cyclins)
• SUMOylation: attachment of a small ubiquitin-like modifier (SUMO) protein to lysine residues (similar to ubiquitination)
  • Example: Many regulators of the cell cycle are regulated via SUMOylation.
  • Opposite: deSUMOylation
• ADP-ribosylation: transfer of an ADP-ribose residue from NAD⁺ by ADP-ribosyltransferase (e.g., histone modification by poly ADP-ribosylation)
• Biotinylation: attachment of biotin (e.g., pyruvate carboxylase uses a biotin cofactor to catalyze the ATP-dependent carboxylation of pyruvate to oxaloacetate)
• Carboxylation: attachment of a carboxylic acid group (R–COOH) (e.g., vitamin K-dependent carboxylation is involved in liver synthesis of clotting factors that bind Ca²⁺)
• Hydroxylation: attachment of hydroxy groups (-OH), typically to proline and sometimes lysine (e.g., important for collagen synthesis to ensure crosslinking outside the cell )
• Methylation: attachment of methyl groups

Irreversible covalent alterations

• Citrullination
  • Posttranslational conversion of the amino acid arginine into citrulline, which is relatively more hydrophobic
  • Carried out by peptidylarginine deiminases

Protein trimming

• Description: the excision of N-terminal or C-terminal propeptides to create a mature protein from an inactive state (e.g., to activate trypsinogen into trypsin)
• Examples
  • Zymogens (e.g., endoproteases)
  • Insulin (in Golgi vesicles)
  • Collagen after secretion

Protein transportation

A protein's intended final destination depends on its signal sequence (if it has one) at the N-terminus and determines if translation is concluded on free ribosomes or ribosomes on the rough ER.

Translocation of ribosomes on the rough ER

Overview

• Proteins that leave the cell (secretory proteins), as well as membrane and lysosomal proteins, are initially synthesized on free ribosomes of the cytosol.
• However, their synthesis is paused shortly after starting and the ribosome is transported to the cytosolic side of the rough ER.
• Protein synthesis then recommences and the protein is directly synthesized into the ER lumen.

Mechanism

1. Initiation of translation on the free ribosomes in the cytosol
2. If a signal sequence (specific amino acid sequence of 9–12 amino acids) is synthesized, it is bound to a signal recognition particle (SRP, a cytosolic ribonucleoprotein ; ).
3. SRP induces a pause in translation and transports the ribosome with the peptide chain (polypeptide-ribosome complex) across the ER membrane.
4. SRP facilitates binding of the ribosome with the signal peptide to the SRP receptor on the ER membrane.
5. SRP and the SRP receptor are both bound to GTP, which is hydrolyzed to GDP; SRP is released and can bind to a new signal sequence.
6. The ribosome is transferred to a translocon, a protein-lined channel composed of a complex of proteins spanning the ER membrane, with opening of the translocon channel. This translocon is termed the sec61 channel.
7. Translation resumes and the protein is synthesized in the ER lumen.
8. The signal sequence is cut off from the growing protein by a signal peptidase.
9. After termination of translation, the ribosome is released into the cytosol.
10. The translocon channel closes and the synthesized protein is left in the ER. During translation, the protein is folded into its native conformation.

If the SRP is absent or dysfunctional, there will be an accumulation of proteins in the cytosol of the cell!

Protein modification and distribution

• Protein modification in the ER
  • N-linked glycosylation
  • Formation of disulfide bonds
• Protein distribution
  • Proteins destined for the cell membrane are directly anchored in the ER membrane.
    • Process: Hydrophobic segments are usually present in the amino acid sequence, which are then integrated into the ER membrane.
  • Soluble proteins for lysosomes or proteins destined to leave the cell via exocytosis remain following completion of translation until they are transported into the ER lumen.
  • All newly synthesized proteins in the ER are transported via the Golgi network to their target destination by transport vesicles.
  • Lysosomal proteins contain a mannose 6-phosphate residue, which is recognized and bound by the membrane-bound mannose 6-phosphate receptor in the trans-Golgi network and transports the proteins to lysosomes in vesicles.
  • Soluble proteins destined to remain in the ER are labeled by the signal sequence KDEL (encodes the letters of the amino acids lysine-aspartate-glutamate-leucine) on their C-terminus.
  • Only properly folded proteins can leave the ER and travel to their final destination.
    • Otherwise, the proteins are bound to chaperones and transported back into the cytosol where they are degraded.

• Main component: initiation phase of translation (regulation via initiation factors)
• Regulation mechanisms
  • Decreased formation of the ternary complex of eIF2, GTP, and initiating tRNA through phosphorylation of the initiation factor eIF2
    • Phosphorylated eIF2 in the GDP-bound form can no longer be converted to its active GTP-bound form. The ternary complex necessary for initiating translation can no longer be formed and the translation rate is reduced.
      • Example: synthesis of globin in red blood cells
        • If there are insufficient levels of heme available to be incorporated in the globin protein, globin synthesis is inhibited by phosphorylation of eIF2.
  • Regulation of the cap recognition process by eIF4