2. bbm translasi biomedik
TRANSCRIPT
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Department of Biochemistry and Molecular Biology School of Medicine, Tarumanagara University
October 2010
The Genetic Code &
Translation Process (Protein Biosynthesis)
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The Genetic Code
• The genetic code is the set of rules by which information encoded in genetic material (DNA or RNA sequences) is translated into proteins (amino acid sequences) by living cells.
• mRNA molecules have no affinity for amino acids and, therefore, that the translation of the information in the mRNA nucleotide sequence into the amino acid sequence of a protein requires an intermediate adapter molecule.
• This adapter molecule (tRNA) must recognize a specific nucleotide sequence on the one hand as well as a specific amino acid on the other.
• Twenty amino acids are required for the synthesis of the cellular proteins; thus, there must be at least 20 distinct codons that make up the genetic code.
• Since there are only 4 different nucleotides in mRNA, each codon must consist of more than a single purine or pyrimidine nucleotide. Codons consisting of two nucleotides each could provide for only 16 (42) specific codons, whereas codons of three nucleotides could provide 64 (43) specific codons.
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• The mRNA transcript is a linear sequence of nucleotides carrying genetic information and it is single-stranded.
• Every three bases of mRNA (a triplet) specifies an amino acid to be added to a growing polypeptide chain; the relationship between the triplets and the corresponding amino acids is the genetic code.
• Each base triplet of mRNA is called a codon. The genetic code is nearly universal for all forms of life.
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Features of the Genetic Code
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MARSHALL Nirenberg (1961)
Relationship between mRNA and the Protein Product
• The order of the codons in the mRNA determines the sequence in which amino acids are added to the growing polypeptida chain.
• The order of the codons in the mRNA determines the linear sequence of amino acids in the protein.
TYPE OF MUTATIONSTYPE Description Example
Point > A Single base changeSilent- > A change that specifies the same amino CGA .-> CGG Arg->ArgMissense- > Different Amino CGA->CCA
Arg -> ProNonsense - >Produce stop kodon CGA->UGA
Arg > StopInsertion- > Addition one or more basesDeletion- > Loss one or more bases
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Translation of mRNA (Protein Synthesis)
• Translation is the process by which the nucleotide sequence of mRNA is converveted to the amino acid sequence of a polypeptide.
• In the first step of the process, all the components needed for translation come together.
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mRNA
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(Kozak Scanning)
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Amino acid activation
aminoacyl-tRNA synthetase
Formation of aminoacyl-tRNA
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The genetic code is translated by means of two sequential "adaptors". The first adaptor is the aminoacyltRNA synthetase enzyme, which couples a particular amino acid to its corresponding tRNA; the second adaptor is the tRNA molecule, whose anticodon forms base pairs with the appropriate nucleotide sequence (codon) on the mRNA. An error in either step will cause the wrong amino acid to be incorporated into a protein chain.
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The incorporation of an amino acid into a protein. A polypeptide chain grows by the stepwise addition of amino acids to its carboxyl-terminal end. The formation of each peptide bond is energetically favorable because the growing carboxyl terminus has been activated by the covalent attachment of a tRNA molecule.
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Decoding an mRNA molecule Each amino acid added to the growing end of a polypeptide chain is selected by complementary base-pairing between the anticodon on its attached tRNA molecule and the next codon on the mRNA chain.
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A comparison of the structures of procaryotic and eucaryotic ribosomes
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The elongation phase of protein synthesis on a ribosome
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Kinetic proofreading selects for the correct tRNA molecule on the ribosome
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The final phase of protein synthesis: The binding of release factor to stop codon terminate translation
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The initiation phase of protein synthesis in eucaryotes
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Recognition of the codon by the anticodon
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Translation initiation pathway in E. coli ribosom
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Translational initiation pathway in eukaryotes.
Initiation factors are represented by colored rectangles where they are first implicated in the pathway and by circles of the same color thereafter. .
Several of the initiation factors: (4A, 4B, 4E, 4G, and 4H) have been left out of the 48S initiation complex for clarity
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• The flow of genetic information follows the sequence DNA → RNA → protein. • The genetic information in the structural region of a gene is transcribed into an RNA molecule such that the sequence of the latter is complementary to that in the DNA.
• Ribosomal RNA (rRNA), transfer RNA (tRNA), and messenger RNA (mRNA), are directly involved in protein synthesis; miRNAs regulate mRNA function at the level of translation and/or stability.
• The information in mRNA is in a tandem array of codons, each of which is 3 nt long.
• mRNA is read continuously from a start codon (AUG) to a termination codon
• The open reading frame, or ORF, of the mRNA is the series of codons, each specifying a certain amino acid, that determines the precise aa sequence of the protein.
• Protein synthesis, like DNA and RNA synthesis, follows the 5' to 3' polarity of mRNA and can be divided into three processes: initiation, elongation, and termination.
• Mutant proteins arise when single-base substitutions result in codons that specify a different amino acid at a given position, when a stop codon results in a truncated protein, or when base additions or deletions alter the reading frame, so different codons are read.
• A variety of compounds, including several antibiotics, inhibit protein synthesis by affecting one or more of the steps involved in protein synthesis.
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Inhibitors of Protein or RNA Synthesis
Acting Only on Procaryotes*
• Tetracycline blocks binding of aminoacyl-tRNA to A-site of ribosome• Streptomycin prevents the transition from initiation complex to chain-elongating ribosome and also causes miscoding.• Chloramphenicol blocks the peptidyl transferase reaction on ribosomes• Erythromycin blocks the translocation reaction on ribosomes• Rifamycin blocks initiation of RNA chains by binding to RNA polymerase (prevents RNA synthesis)
Acting Only on Procaryotes and Eucaryotes
• Puromycin causes the premature release of nascent polypeptide chains by its addition to growing chain end.• Actinomycin D binds to DNA and blocks the movement of RNA polymerase (prevents RNA synthesis)
Acting Only on Eucaryotes
• Cycloheximide blocks the translocation reaction on ribosomes.• Anisomycin blocks the peptidyl transferase reaction on ribosomes• α-Amanitin blocks mRNA synthesis by binding preferentially to RNA polymerase II
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REGULASI EKSPRESI GEN
• Ekspresi gen merupakan keseluruhan proses, di mana informasi genetik yang disimpan dalam DNA diubah menjadi produk fungsional gen yaitu RNA atau protein.
• Tujuan regulasi ekspresi gen adalah agar ekspresinya terjamin secara akurat baik spatial maupun temporal, sehingga di satu pihak sel dapat beradaptasi terhadap perubahan lingkungan, sedangkan di pihak lain sel dapat tumbuh dan berdiferensiasi secara normal.
• Untuk mencapai tujuan tersebut , regulasi ekspresi gen terjadi dalam berbagai tingkat.
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(1) controlling when and how often a given geneis transcribed (transcriptional control), (2) controlling how the primary RNA transcript is spliced or otherwise processed (RNA processing control), (3) selecting which completed mRNAs in the cell nucleus are exported to the cytoplasm (RNA transport control), (4) selecting which mRNAs in the cytoplasm are translated by ribosomes (translational control), (5)
selectively destabilizing certain mRNA molecules in the cytoplasm (mRNA degradation control) (6) selectively activating, inactivating, or compartmentalizing specific protein molecules after they
have been made (protein activity control)
REGULASI EXPRESSI GENE
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Polypeptide Synthesis: An Overviewa. Polypeptide Synthesis Proceeds from N-Terminus to C-Terminus (Howard Dintzis, (1961)
b. Ribosomes Read mRNA in the 5’ → 3’ direction
C. Active Translation Occurs on Polyribosomes
d. Chain Elongation Occurs by the Linkage of the Growing Polypeptide to the Incoming tRNA’s Amino Acid Residue
Chain Initiation a. fMet Is the N-Terminal Residue of Prokaryotic Polypeptides
b. Base Pairing between mRNA and the 16S rRNA Helps Select the Translational Initiation Site
c. Prokaryotic Initiation Is a Three-Stage Process that Requires the Participation of Soluble Protein Initiation Factors
d. Eukaryotic Initiation Is Far More Complicated than That of Prokaryotes
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Translational initiation pathway in eukaryotes.
Initiation factors are represented by colored rectangles where they are first implicated in the pathway and by circles of the same color thereafter.
The higher order complexes are hypothetical.
Several of the initiation factors: (4A, 4B, 4E, 4G, and 4H) have been left out of the 48S initiation complex for clarity
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POSTTRANSLATIONAL MODIFICATION
B. Signal Peptides Are Removed from Nascent Proteins by a Signal Peptidase
A. Proteolytic Cleavage
C. Polyproteins
D. Covalent Modification: - Collagen Assembly Requires Chemical Modification
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• Nitrosylation• Glycation• ADP-ribosylation• O-GlcNAc• Lipidation, ex:- Glycophosphatidyl inositol (GPI) membrane protein
- Prenyl groups, Farnesylation
Additional PTMs
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Protein Folding
• Protein disulfide isomerase (PDI)
• Molecular Chaperones:
• heat shock proteins (Hsp), ex: HSP70• Chaperonins:
• Hsp60 proteins (GroEL in E. coli)• Hsp10 proteins (GroES in E. coli),
The X-ray structure of the GroEL–GroES–(ADP)7 chaperonin complex
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Hsp70 is believed to bind and stabilize nascent polypeptides early in their synthesis--preventing misfolding and aggregation
Hsp70 binding and release, in an ATP-dependent manner, may help proteins fold to the native state OR Hsp70 may ‘transfer’ non-native proteins to other chaperones for folding (e.g., chaperonins)
Hsp70 is also important during cellular stresses (thermotolerance), and has numerous other functions in the cell apart from assisting de novo protein folding. It often works in collaboration with other chaperones, especially Hsp40
Hsp70 in Hsp70 in de novode novo protein biogenesis protein biogenesis