Translation (Protein Synthesis) · During Protein Synthesis Attachment of Amino acid to tRNA is the...

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Translation (Protein Synthesis) Aung Win Tun Department of Biochemistry Faculty of Medicine Siriraj Hospital Mahidol University [email protected]

Transcript of Translation (Protein Synthesis) · During Protein Synthesis Attachment of Amino acid to tRNA is the...

Page 1: Translation (Protein Synthesis) · During Protein Synthesis Attachment of Amino acid to tRNA is the critical step for fidelity of genetic information. Formation of Aminoacyl tRNA

Translation (Protein Synthesis)

Aung Win Tun

Department of Biochemistry

Faculty of Medicine Siriraj Hospital

Mahidol University

[email protected]

Page 2: Translation (Protein Synthesis) · During Protein Synthesis Attachment of Amino acid to tRNA is the critical step for fidelity of genetic information. Formation of Aminoacyl tRNA

Central Dogma of Life

DNA

RNA

Protein

transcription

translation

Page 3: Translation (Protein Synthesis) · During Protein Synthesis Attachment of Amino acid to tRNA is the critical step for fidelity of genetic information. Formation of Aminoacyl tRNA

Central Dogma of Life

DNA

RNAs

Protein

transcription

translation

mRNA, tRNA, rRNA

Post transcriptional modification

Page 4: Translation (Protein Synthesis) · During Protein Synthesis Attachment of Amino acid to tRNA is the critical step for fidelity of genetic information. Formation of Aminoacyl tRNA

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.

Page 5: Translation (Protein Synthesis) · During Protein Synthesis Attachment of Amino acid to tRNA is the critical step for fidelity of genetic information. Formation of Aminoacyl tRNA

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

Page 6: Translation (Protein Synthesis) · During Protein Synthesis Attachment of Amino acid to tRNA is the critical step for fidelity of genetic information. Formation of Aminoacyl tRNA

The translation of the 4 letter alphabets of nucleic acids into the amino acids sequence of proteins.

mRNA

Proteins

Page 7: Translation (Protein Synthesis) · During Protein Synthesis Attachment of Amino acid to tRNA is the critical step for fidelity of genetic information. Formation of Aminoacyl tRNA

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

Page 8: Translation (Protein Synthesis) · During Protein Synthesis Attachment of Amino acid to tRNA is the critical step for fidelity of genetic information. Formation of Aminoacyl tRNA

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)

Page 9: Translation (Protein Synthesis) · During Protein Synthesis Attachment of Amino acid to tRNA is the critical step for fidelity of genetic information. Formation of Aminoacyl tRNA

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?

Page 10: Translation (Protein Synthesis) · During Protein Synthesis Attachment of Amino acid to tRNA is the critical step for fidelity of genetic information. Formation of Aminoacyl tRNA

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

Page 11: Translation (Protein Synthesis) · During Protein Synthesis Attachment of Amino acid to tRNA is the critical step for fidelity of genetic information. Formation of Aminoacyl tRNA

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)

Page 12: Translation (Protein Synthesis) · During Protein Synthesis Attachment of Amino acid to tRNA is the critical step for fidelity of genetic information. Formation of Aminoacyl tRNA

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.

Page 13: Translation (Protein Synthesis) · During Protein Synthesis Attachment of Amino acid to tRNA is the critical step for fidelity of genetic information. Formation of Aminoacyl tRNA
Page 14: Translation (Protein Synthesis) · During Protein Synthesis Attachment of Amino acid to tRNA is the critical step for fidelity of genetic information. Formation of Aminoacyl tRNA

Initiation

Page 15: Translation (Protein Synthesis) · During Protein Synthesis Attachment of Amino acid to tRNA is the critical step for fidelity of genetic information. Formation of Aminoacyl tRNA

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

Page 16: Translation (Protein Synthesis) · During Protein Synthesis Attachment of Amino acid to tRNA is the critical step for fidelity of genetic information. Formation of Aminoacyl tRNA

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.

Page 17: Translation (Protein Synthesis) · During Protein Synthesis Attachment of Amino acid to tRNA is the critical step for fidelity of genetic information. Formation of Aminoacyl tRNA
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Elongation

P site – Peptidyl siteA site – aminoacyl siteE site – exit

1. Position the aminoacyl-tRNA to A site

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Elongation

2. Formation of peptide bondby Peptidyl Transferase

Chloramphenicol inhibits peptidyl transferase.

Page 20: Translation (Protein Synthesis) · During Protein Synthesis Attachment of Amino acid to tRNA is the critical step for fidelity of genetic information. Formation of Aminoacyl tRNA

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

Page 21: Translation (Protein Synthesis) · During Protein Synthesis Attachment of Amino acid to tRNA is the critical step for fidelity of genetic information. Formation of Aminoacyl tRNA

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.

Page 22: Translation (Protein Synthesis) · During Protein Synthesis Attachment of Amino acid to tRNA is the critical step for fidelity of genetic information. Formation of Aminoacyl tRNA

Termination

Termination codon appears in the A site.No corresponding tRNA for termination codon.

Page 23: Translation (Protein Synthesis) · During Protein Synthesis Attachment of Amino acid to tRNA is the critical step for fidelity of genetic information. Formation of Aminoacyl tRNA

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.

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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

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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.

Page 26: Translation (Protein Synthesis) · During Protein Synthesis Attachment of Amino acid to tRNA is the critical step for fidelity of genetic information. Formation of Aminoacyl tRNA

Prokaryotes have no post transcriptional modification of mRNA and no distinct nuclear compartment. Translation occurs while transcription is going on.

Page 27: Translation (Protein Synthesis) · During Protein Synthesis Attachment of Amino acid to tRNA is the critical step for fidelity of genetic information. Formation of Aminoacyl tRNA

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

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Synthesis of Export Proteins: Signal Hypothesis

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Synthesis of ……

Secretory proteins (Export Proteins)Proteins of Plasma MembraneProteins of Membrane of Endoplasmic ReticulumLysosomal Proteins

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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

Page 31: Translation (Protein Synthesis) · During Protein Synthesis Attachment of Amino acid to tRNA is the critical step for fidelity of genetic information. Formation of Aminoacyl tRNA

Properties of Signal Sequence

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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.

Page 34: Translation (Protein Synthesis) · During Protein Synthesis Attachment of Amino acid to tRNA is the critical step for fidelity of genetic information. Formation of Aminoacyl tRNA

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.

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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.

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Rough Endoplasmic Reticulum