The Central Dogma of Molecular Biology
Transcription
Macromolecule
Basic Units
DNA
Nucleic acids
Features of the Genetic Code
- code of nucleotide triplets
- degenerate but unambiguous
- almost universal
- codons (nucleotide triplets) are
non-overlapping and follow one
another consecutively.
Translation
RNA
protein
Amino acids
Participants in Translation
Messenger RNA – structure
Transfer RNA – structure and aminoacylation
The ribosome – structure
Mechanisms of Translation
Initiation
Elongation
Termination
Translation and Antibiotics
Translation in Eukaryotes
mRNA Structure - Prokaryotes
mRNA Features
- polycistronic mRNAs (operons)
- co-regulated genes occur in operons
tRNA – Structure and Aminoacylation
- tRNAs are the interpreters of the genetic code.
- common secondary and tertiary structure.
- tRNA bases are highly modified.
-L shaped molecules created by coaxial stacking of helices
and a network of tertiary interactions.
Crick’s Wobble Hypothesis
Aminoacylation
- catalyzed by aminoacyl
tRNA synthetases.
- two step reaction.
- high fidelity achieved by
proofreading (2nd step).
Ribosome Structure
- major constituents of the cell, 25% dry mass.
- highly conserved protein & RNA components.
- 1.5 MD Mol. Wt.
5.5 Å Resolution of 70S particle
from Thermus thermophilus.
Science 292, 883-896, 2001.
Mechanisms of Translation
Initiation
- binding of initiation factors, IF1, IF2-GTP &
IF3 to free 30S subunit confers ability to bind
mRNA and initiating aminoacyl tRNA.
- binding of Shine-Dalgarno sequence to 3’ end
of 16S rRNA aligns AUG, allows binding of
initiator tRNA and dissociates IF3.
- release of IF3 creates a complex with high
affinity for binding the 50S subunit. A, P & E.
- 50S binding stimulates hydrolysis of IF2-GTP
to IF2-GDP and leads to dissociation of IFs.
Elongation
- binding of aminoacyl tRNA-EFTuGTP
to A site. Binding stimulates hydrolysis
and release of EFTu-GDP.
- regeneration of EFTu-GTP is achieved
with the help of EF-Ts (guanine n’tide
exchange factor.
- peptide bond formation (peptidyl
transferase activity provided by 23S
rRNA.
-translocation of peptidyl tRNA to P
site with hydrolysis of GTP by EF-G.
Termination
- signaled by one of three termination codons.
- binding of RF1 (UAA, UAG) or RF2 (UAA
& UGA) to the A site and binding of RF3GTP.
- peptidyl transfer to a water molecule in A site
with GTP hydrolysis.
- release of protein, dissociation of P site tRNA,
release factors and the 50S subunit.
-mRNA may dissociate or remain bound to 30S
subunit for initiation at downstream AUGs.
Energetics of Peptide Bond Formation
Protein synthesis carries a high energetic cost.
For a protein N residues in length:
N moles of ATP for charging tRNAs
1 mole of GTP for initiation
N-1 moles of GTP for peptide bond formation
N-1 moles of GTP for translocation
1 mole of GTP for termination
3N moles of high energy phosphate molecules hydrolyzed
Synthesis of a typical protein of 300 amino acids, where each high energy phosphate yields
~ 40 kJ/mole requires the expenditure of 36,000 kJ of energy (8,600 kcal).
Expressed in terms of energy per peptide bond formed: 120 kJ (29 kcal). Since the energy
of hydrolysis of a peptide bond is only about -2 kJ, the cost of synthesis appears exorbitant!
The reason - synthesis of a specific sequence rather than a random sequence (20300 ≅ 10390)
carries a very high entropic penalty (it takes energy to create order!)
Translation and Antibiotics
Translation in Eukaryotes