Translation (Protein Synthesis)
Aung Win Tun
Department of Biochemistry
Faculty of Medicine Siriraj Hospital
Mahidol University
Central Dogma of Life
DNA
RNA
Protein
transcription
translation
Central Dogma of Life
DNA
RNAs
Protein
transcription
translation
mRNA, tRNA, rRNA
Post transcriptional modification
Aminoacyl tRNA Synthetase Charging of amino acids / Activation of amino acids
Amino acid is attached to the specific tRNA specific aminoacyl tRNA synthetase
tRNA Aminoacyl- tRNA
chargingDuring Protein Synthesis
Attachment of Amino acid to tRNA is the critical step for fidelity of genetic information.
Formation of Aminoacyl tRNAAminoacyl tRNA Synthetase Charging of amino acids / Activation of amino acids Amino acid is attached to the specific tRNA specific aminoacyl tRNA
synthetase 20 different synthetases for 20 different amino acids Proof-reading and editing activity that can remove mischarged amino acids
from tRNA
The translation of the 4 letter alphabets of nucleic acids into the amino acids sequence of proteins.
mRNA
Proteins
5’ …………..CCA CAG AGU GGU GAC UUU…….. 3’
3’ …………..GGU GUC TCA CCA CUG AAA……. 5’5’ …………..CCA CAG AGT GGU GAC TTT…….. 3’
………….. Pro Gln Ser Gly Asp Phe……..
Transcription
Translation (Protein Synthesis)
mRNA directs the ordered polymerization of specific amino acids!
N terminus C terminus
1. mRNA carries genetic information copied from DNA. (messenger for
information)
2. rRNAs together with proteins form ribosome provide site for protein synthesis.
3. tRNA carries amino acids required for protein synthesis. (Adaptor molecule
between code and amino acid)
The sequence of codons that runs from a specific start codon to a stop codon is called a reading frame. (ORF)
mRNA 5’ 3’5’ UTR 3’ UTR
UTR = untranslated region
Exon
Do all the nucleotides in mRNA code for amino acids of proteins?
The sequence of codons that runs from a specific start codon to a stop codon is called a reading frame. (ORF)
mRNA 5’ 3’5’ UTR 3’ UTR
UTR = untranslated region
Exon
Start codon Stop codon
Initiation1. mRNA
2. Met-tRNAi (Eukaryotes)
fMet-tRNA (formyl Met-tRNA in Prokaryotes)
1. Ribosome
2. Initiation Factors (IF/eIF)
3. GTP
4. ATP (during the aminoacylation/charging)
The initiating AUG is guided to its correct position by the Shine-Dalgarno sequence in the mRNA.
Shine-Dalgarno sequence is four to nine purine residues, 8 to 13 bp to the 5’ side of the initiation codon.
Initiation
The first amino acid to be incorporated in Prokaryotes is formyl-Met (fMet).IF2 only recognizes fMet, not other Met.IN Eukaryotes, it is met-tRNA.
Initiation
Initiation
P site – Peptidyl siteA site – aminoacyl siteE site – exit
StreptomycinStreptomycin – binds 30 S subunit and distort the structure of assemblinginitiation complex.TetracyclineTetracycline - binds the 30S ribosomal subunit, thereby blocking the assess of the aminoacyl-tRNA to the mRNA ribosomal complx.Aminoglycosides (Gentamycin)Aminoglycosides (Gentamycin) - binds 30S ribosomal subunit, interfering the assemply of the functional ribosomal apparatus.
Elongation
P site – Peptidyl siteA site – aminoacyl siteE site – exit
1. Position the aminoacyl-tRNA to A site
Elongation
2. Formation of peptide bondby Peptidyl Transferase
Chloramphenicol inhibits peptidyl transferase.
3. Translocation by Translocase Then, A site is free and ready to accept incoming am-tRNA. The process will continue till termination codon is reached.
Elongation
3. Translocation by Translocase Then, A site is free and ready to accept incoming am-tRNA. The process will continue till termination codon is reached.
ElongationErythromycin and clindamycin bind 50 S subunit , inhibit translocation step.
Diptheria toxins – (Corynebacterium diphtheriae) causes ADP-ribosylation of eEF-2, inhibiting translocation in
Eukaryotes.
Termination
Termination codon appears in the A site.No corresponding tRNA for termination codon.
Post Translational Modification
1. Removal of some amino acids (trimming)Removal of amino terminal residues.
2. Covalent modificationPhosphorylation – on threonine, serine, tyrosine
phosphorylation of milk protein CaseinGlycosylation – carbohydrate chains attached to serine or threonine or
amide nitrogen of asparagine Hydroxylation - on Proline and Lysine of collagen Carboxylation – clotting factor prothrombin
After its release from the ribosome, a newly synthesized protein folds into its native three-dimensional conformation, a process facilitated by other proteins called chaperones.
Genetic message of mRNA >>>> 3-D structure of the protein
In both prokaryotes and Eukaryotes, the newly synthesized proteins do not attain their final biologically active conformation until they have been altered by one or more modifications.
Post Translational Modification
3. Addition of prosthetic group
Addition of heme group to Haemoglobin
4. Proteolytic processing
Proinsulin >>> insulin
Trypsinogen >>> Trypsin
5. Disulfide bond formation
Between Cystein residues
Polysome or Polyribosome
More than one ribosome attached to a single mRNA at any given time.
Polysome - complex of mRNA with multiple ribosomes
Each ribosome carries a nascentpolypeptide chain that grows longer as the ribosome approaches the 3’ end of mRNA.
Prokaryotes have no post transcriptional modification of mRNA and no distinct nuclear compartment. Translation occurs while transcription is going on.
Polycistronic mRNA
- A single mRNA contains information for more than one proteins.
- In Prokaryotes
Monocistronic mRNA
- A single mRNA contains information for only one protein.
- In Eukaryotes
Synthesis of Export Proteins: Signal Hypothesis
Synthesis of ……
Secretory proteins (Export Proteins)Proteins of Plasma MembraneProteins of Membrane of Endoplasmic ReticulumLysosomal Proteins
Signal Peptide to mediate attachment of ribosome to Endoplasmic Reticulum
Newly synthesized protein Newly synthesized proteinwith signal sequence
Free ribosome Membrane bound ribosome
NH3 NH3
Mediate attachment to ER
Properties of Signal Sequence
1, 2 Once the signal sequence emerges from the ribosome, it is bound by a signal-recognition particle (SRP).
3 The SRP delivers the ribosome/nascent polypeptide complex to the SRP receptor in the ER membrane. This interaction is strengthened by binding of GTP to both the SRP and its receptor.
4 Transfer of the ribosome/nascent polypeptide to the translocon leads to opening of this translocation channel and insertion of the signal sequence and the growing polypeptide into the central pore.Both the SRP and SRP receptor, once dissociated from the translocon, hydrolyze their bound GTP and then are ready to initiate the insertion of another polypeptide chain.
5 As the polypeptide chain elongates, it passes through the translocon channel into the ER lumen, where the signal sequence is cleaved by signal peptidase and is rapidly degraded.
6The peptide chain continues to elongate as the mRNA is translated toward the 3’ end. Because the ribosome is attached to the translocon, the growing chain is extruded through the translocon into the ER lumen.
7,8Once translation is complete, the ribosome is released, the remainder of the protein is drawn into the ER lumen, the translocon closes, and the protein assumes its native folded conformation.
The proteins travels in vesicles to The Golgi, where it may be glycosylatedfurther and is packaged in the secretory vesicles.
Secretory vesicles containing the proteinstravel from the Golgi to the cell membrane.The protein is released from the cell byexocytosis.
Rough Endoplasmic Reticulum