Molecular Biology of Translation: Initiation, Elongation, and Post-Translational Modifications

Initiation complex

mRNA ribosome (70S in prokaryotes or 80S in eukaryotes) and initiator tRNA carrying fMet in prokaryotes or Met in eukaryotes

Aminoacyl-tRNAs

tRNAs carrying specific amino acids attached at their 3' end and possessing an anticodon complementary to the mRNA codon

Charged tRNA

A tRNA that has an amino acid covalently attached; uncharged tRNAs lack an amino acid

Aminoacyl-tRNA synthetase

Enzyme that activates and attaches an amino acid to its specific tRNA molecule

Energy source for amino acid activation

ATP hydrolysis provides energy to attach amino acids to tRNAs

Intermediate formed during amino acid activation

Aminoacyl-AMP formed when amino acid reacts with ATP releasing pyrophosphate

Attachment site of amino acid on tRNA

The 3'-hydroxyl group of the ribose on the terminal adenine nucleotide at the 3' end of the tRNA

Class I aminoacyl-tRNA synthetase

Attaches the amino acid to the 2'-OH of ribose then transesterifies to 3'-OH

Class II aminoacyl-tRNA synthetase

Attaches the amino acid directly to the 3'-OH of ribose

Active form of tRNA for translation

3'-aminoacyl ester form because only 3'-esters are activated substrates for protein synthesis

Number of aminoacyl-tRNA synthetases

20 total one for each amino acid

Role of aminoacyl-tRNA synthetase in accuracy

Ensures the correct amino acid is attached to the correct tRNA maintaining fidelity of translation

Codon-anticodon pairing

Anticodon on tRNA pairs antiparallel to mRNA codon; codon read 5'→3' and anticodon read 3'→5'

Wobble hypothesis

First two codon-anticodon base pairs follow Watson-Crick rules; the third codon base can pair noncanonically allowing flexibility

Purpose of wobble pairing

Allows one tRNA to recognize multiple codons for the same amino acid increasing efficiency

Requirements for elongation

Initiation complex aminoacyl-tRNA elongation factors and GTP

Elongation factor delivering aminoacyl-tRNA in prokaryotes

EF-Tu bound to GTP

Eukaryotic equivalent of EF-Tu

eEF1A bound to GTP

Energy change during aminoacyl-tRNA delivery

GTP hydrolysis to GDP and Pi provides energy for tRNA placement in the A-site

Catalyst for peptide bond formation

Peptidyl-transferase activity of the large ribosomal subunit's rRNA (23S in prokaryotes 28S in eukaryotes)

Type of enzyme peptidyl-transferase is

Ribozyme because the catalytic activity is carried out by rRNA not protein

Bond formed during elongation

Peptide bond between carboxyl group of P-site amino acid and amino group of A-site amino acid

Action after peptide bond formation

Ribosome translocates three nucleotides toward the 3' end of the mRNA

Elongation factor driving translocation in prokaryotes

EF-G bound to GTP

Eukaryotic equivalent of EF-G

eEF2 bound to GTP

tRNA movement during translocation

Uncharged tRNA moves from P-site to E-site and exits while peptidyl-tRNA moves from A-site to P-site

Ribosome site empty after translocation

A-site opens for next aminoacyl-tRNA

Translation termination trigger

Stop codon (UAA UAG or UGA) enters A-site

Prokaryotic release factors

RF1 recognizes UAA and UAG RF2 recognizes UAA and UGA RF3 is a GTPase assisting peptide release

Eukaryotic release factors

eRF1 recognizes all three stop codons and eRF3 (GTPase) assists peptide release

Event when release factor binds stop codon

Peptidyl-transferase hydrolyzes bond between polypeptide and tRNA releasing completed protein

Fate of ribosome after termination

Ribosomal subunits dissociate from mRNA and each other ready for reuse

Polysomes (polyribosomes)

Structures with multiple ribosomes translating a single mRNA simultaneously increasing efficiency

Function of polysomes

Allow rapid production of many copies of a protein from one mRNA strand

Polysomes in prokaryotes

Translation begins before transcription finishes due to coupled transcription-translation

Polysomes in eukaryotes

Found free in cytoplasm for cytosolic proteins or bound to rough ER for secretory and membrane proteins

Energy source for amino acid activation

ATP hydrolysis (ATP → AMP + PPi)

Energy source for elongation and translocation

GTP hydrolysis by EF-Tu and EF-G (or eEF1A and eEF2)

Direction of mRNA reading and protein synthesis

mRNA read 5'→3'; polypeptide grows from N-terminus to C-terminus

Post-translational modifications

Chemical changes to a polypeptide after synthesis that affect function stability localization and activity

Types of post-translational modifications

Proteolytic cleavage phosphorylation glycosylation acetylation methylation ubiquitination and formation of disulfide bonds

Proteolytic cleavage

Removal of N-terminal methionine or signal peptide and cleavage of pro-peptides to activate enzymes or hormones

Phosphorylation

Addition of phosphate group (by kinases) to serine threonine or tyrosine residues regulating activity or signaling

Glycosylation

Addition of carbohydrate chains to asparagine (N-linked) or serine/threonine (O-linked) residues important for folding stability and secretion

Acetylation

Addition of acetyl groups to lysine residues or N-termini affecting stability localization and gene regulation

Methylation

Addition of methyl groups to lysine or arginine residues influencing protein-protein interactions and epigenetic regulation

Ubiquitination

Covalent attachment of ubiquitin marking proteins for degradation in the proteasome

Disulfide bond formation

Covalent bonds between cysteine residues stabilizing tertiary or quaternary structure of secreted proteins

Protein targeting

Process that directs newly synthesized proteins to correct cellular or extracellular locations

Signal peptide

Short N-terminal amino acid sequence that directs ribosome to rough ER for secretion or membrane insertion

Cytoplasmic proteins

Synthesized on free ribosomes and remain in cytoplasm

Secretory and membrane proteins

Synthesized on rough ER ribosomes and enter the ER lumen during translation

Signal recognition particle (SRP)

Binds signal peptide and ribosome halts translation until complex docks on SRP receptor on ER membrane

SRP receptor and translocon

Facilitate ribosome docking and insertion of growing polypeptide into ER lumen or membrane

Cleavage of signal peptide

Signal peptidase removes signal sequence after entry into ER

Protein folding in ER

Assisted by chaperone proteins and formation of disulfide bonds

Protein sorting after ER

Proteins move to Golgi for further modification and sorting to lysosomes plasma membrane or secretion

Targeting of mitochondrial and chloroplast proteins

Directed by N-terminal transit peptides recognized by import receptors on organelle membranes

Nuclear localization signal (NLS)

Sequence directing proteins to the nucleus via nuclear pore transport

Peroxisomal targeting signal (PTS)

C-terminal or N-terminal sequence targeting proteins to peroxisomes

Protein degradation pathways

Ubiquitin-proteasome system for short-lived proteins and lysosomal degradation for long-lived or extracellular proteins

Proteasome function

Multisubunit complex that recognizes ubiquitinated proteins and degrades them into peptides using ATP