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 learning card.
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
- 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
- Unambiguity (genetic code): codon is specific to only 1 AA.
Non-overlapping (genetic code): Each codon of the mRNA is translated in the 5' to 3' direction continuously in an open reading frame (ORF), beginning with the start codon and ending with the stop codon.
- Exceptions: some viruses
Degeneracy (genetic code): The genetic code is redundant, meaning most AAs are encoded by > 1 codon.
- Exceptions: methionin and tryptophan
- Reduces the extent of damage caused by DNA mutations.
- Due to tRNA wobble: Some tRNA molecules are able to recognize multiple codons through a certain degree of flexibility (wobble) between the pairing of the third nucleotide of the codon with the first nucleotide of the anticodon.
- Universality (genetic code): Almost all organisms use the same genetic code (has not evolved).
To help remember the stop codons UGA, UAG, and UAA: U Go Away, U Are Gone, and U Are Away!
- Description: binding of tRNA to its corresponding AA
- Reaction mechanism: The charging of tRNA in the cytosol occurs in two steps and is catalyzed by aminoacyl tRNA synthetases.
- Mischarged tRNA:
Ribosome binding sites
- See “Ribosomes” in the cell for more details.
- Ribosome is composed of rRNA and proteins that form subunits:
- Binding site for mRNA is on the small ribosomal subunit.
- Both ribosomal subunits jointly form the binding sites for tRNA.
- Because of the length of most mRNA, more than one ribosome can bind them, allowing synthesis of multiple polypeptides at once = polysome.
For binding sites, think of an “APE” party: 1. A site → Arrival with Aminoacyl-tRNA 2. P site → Growing (GTP) Party of Peptides 3. E site → party Ends and is EmptytRNA Exits; Growing stands for GTP as energy source
- 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)
- Initiator met-tRNA, eukaryotic IF2 (eIF2), and GTP bind to the small ribosomal subunit to form a preinitiation complex; (initially a 43s preinitiation complex).
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)
- 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).
- Directions of processes
- 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 between the AAs bound to the A-sites and P-site (via tRNA).
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 unloaded tRNA is released from the E-site.
- The ribosome moves one triplet along the mRNA in the 3' direction.
- A release factor recognizes the stop codon and hydrolytically cleaves the peptidyl tRNA bonds (requires GTP) → release of the protein
- 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.
- The spatial structure is specified in the AA sequence of the protein (see 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
- 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.
- Examples: heat shock proteins (e.g., Hsp70, Hsp60, Hsp90) prevent denaturation or misfolding at high temperatures or when under chemical stress
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.
- Usually a trans confirmation; however, the cis conformation is at times more favorable.
- Description: lack of folding or non-native protein folding as a result of denaturing factors
- Mechanisms of misfolding
- Intracellular reaction: Misfolded proteins are identified and either rescued by chaperones or are ubiquitinated and labeled for proteasomal degradation (see ).
- Consequences of misfolding
Many proteins require specific covalent alterations (co- or post-translational modifications) in addition to correct folding to function properly. Examples include glycosylation, lipid anchors, phosphorylation, acetylation, ubiquitination, ADP-ribosylation, biotinylation, carboxylation, methylation and hydroxylation.
- Enzymatic 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).
|N-linked glycosylation||O-linked glycosylation|
|% of all glycosylations||∼ 90%||∼ 10%|
|Attached by||N-glycosidic bonds||O-glycosidic bonds|
|Amino acids involved||Asparagine|| |
Serine or threonine
|Carbohydrate side chains||Complex oligosaccharides||Less complex oligosaccharides|
|Sugar residues involved|| || |
|Enzymes/molecules involved|| |
N-linked glycosylation, the attachment of sugar to the asparagine residue of proteins, begins in the rough ER! Enzymatic glycosylation should not be mistaken 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.)
- 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.
- 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. Additionally, the durability of proteins may be influenced.
- Phosphorylation: attachment of phosphate residues to the hydroxyl group (OH group) of serine or threonine or of tyrosine residues by kinases
- Acetylation: linkage with a CO-CH3 group by acetyltransferases
- Ubiquitination: attachment of ubiquitin (a small protein) to the ε-amino group of lysine residues in proteins, especially in proteins to be degraded
- SUMOylation: attachment of a small ubiquitin-like modifier (SUMO) protein to lysine residues (similar to ubiquitination)
- ADP-ribosylation: transfer of an ADP-ribose residue from NAD+ by ADP-ribosyltransferase
- Biotinylation: attachment of biotin
- Carboxylation: attachment of a carboxylic acid group (R–COOH)
- Hydroxylation: attachment of hydroxy groups (-OH), typically to proline and sometimes lysine (requires vitamin C)
|Mitochondria, peroxisomes, or nucleus||Yes||Free ribosomes|
|Cell membrane, lysosomes, or extracellular (secretory proteins)||Yes||Rough ER ribosomes|
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.
- Initiation of translation on the free ribosomes in the cytosol
- 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 ribonucleoprotein):
- SRP induces a pause in translation and transports the ribosome with the peptide chain across the ER membrane.
- SRP facilitates binding of the ribosome with the signal peptide to the SRP receptor on the ER membrane.
- 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.
- 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.
- Translation resumes and the protein is synthesized in the ER lumen.
- The signal sequence is cut off from the growing protein by a signal peptidase.
- After termination of translation, the ribosome is released into the cytosol.
- The translocon channel closes and the synthesized protein is left in the ER.
- During translation, the protein is folded into its native conformation.
Protein modification in the ER
- N-linked glycosylation
- Formation of disulfide bonds
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.
- Proteins destined for the cell membrane are directly anchored in the ER membrane.
- Main component: initiation phase of translation (regulation via initiation factors)
- 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.
- Regulation of the cap recognition process by eIF4
- Decreased formation of the ternary complex of eIF2, GTP, and initiating tRNA through phosphorylation of the initiation factor eIF2