Genetics Instructor: Dr. Jihad Abdallah Topic 5: Translation of RNA (Protein synthesis) 1
Central dogma of genetics DNA In the nucleus Transcription mrna Translation In the cytoplasm Protein 2
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The primary role of DNA is to store the information needed for the synthesis of all proteins that an organism can make. The genes that encode an amino acid sequence are called structural genes The RNA that is transcribed from structural genes is called messenger RNA (mrna). During translation the genetic code within mrna is used to make a polypeptide with a specific amino acid sequence. The sequence of bases in mrna is read in groups of three nucleotides known as codons. 4
The sequence of three bases of most codons codes for a particular amino acid. For example, the codon CCC encodes the amino acid proline and GGC encodes for glycine. The codon AUG which encodes for methionine is used as a start codon and the polypeptide sequence starts with methionine. One of three codons can end the process of translation (UAA, UAG, or UGA) and called stop codons (or nonsense codons). These stop codons do not code for amino acids (except that UGA codes for Tryptophan in mammalian mitochondria while UAA and UAG code for Glutamine in ciliated protozoa) Because mrna contains 4 different bases (A, U, C, G) there are 64 different possible codons. 5
Because the number of possible codons exceeds the number of amino acids (20), the genetic code is degenerate. This means that more than one codon can specify the same amino acid. For example, GGU, GGC, GGA, and GGG all encode the amino acid glycine. Transfer RNA (trna) plays a major role in translation of mrna into protein. Each trna contains an anticodon triplet sequence complementary to a specific codon on the mrna and carries the specific amino acid coded for by the mrna codon. The amino acid is charged (loaded) on the trna by the enzyme aminoacyl-trna synthetase. 6
Secondary structure of trna (Cloverleaf model) aa 7
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Translation occurs on the surface of the ribosome. Each ribosome is composed of two subunits (called the large and small subunits). Each subunit itself is formed from the assembly of many different proteins and rrna molecules. Bacterial ribosomes contain two subunits: - 30S: contains 21 different proteins and 16S rrna - 50S: contains 34 proteins, 5S and 23S rrna. Eukaryotic ribosomes found in the cytosol contain two subunits: - 40S: contains 33 proteins and 18S rrna - 60S: contains 49 proteins and 5S, 5.8S and 28S rrnas. Note: the S in 30S, etc represents Svedberg units (a measure of time of sedimentation) 9
Ribosomes contain specific sites where trnas bind and the polypeptide is synthesized. These sites are known as the peptidyl site (P site), the aminoacyl site (A site) and the exit site (E site). Translation occurs in three steps: 1. Initiation 2. Elongation 3. Termination 10
Initiation Peptide synthesis always starts from methionine (Met). The mrna binds to the small subunit of the ribosome with the help of initiation factors (IF). The initiation factors recognise the 7-methylguanosine cap and aid in the identification of the start codon (AUG). The initiator trna which contains the anticodon UAC and carries the amino acid methionine (called trna i met ) binds to the start codon in the P site. The large subunit of the ribosome then associates with the small subunit. This aggregate represents the initiation complex 11
Elongation During this stage, new amino acids are added one at a time forming a polypeptide chain. The newly arrived trna always binds to the A site ("A" for "aminoacyl") adding a new amino acid which bounds to the previous amino acid by a peptide bond catalyzed by peptidyl transferase enzyme which is part of the large subunit. At the same time, the covalent bond between the amino acid and the trna occupying the P site is broken. 12
The uncharged trna exits the ribosome and the ribosome moves one codon (3 bases) down the mrna chain (translocation) causing the trna present in the A site to enter the P site. Then another trna binds the vacant A site and a new peptide bond is formed with the amino acid it carries. Similar steps are repeated in the next cycles of elongation. The activity of peptidyltransferase involves the transfer of the polypeptide from the P site to the A site to form the peptide bond. 13
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Termination Protein synthesis will terminate when the ribosome arrives at one of the three stop codons (UAA, UAG, or UGA). Stop codons do not bind to trna. The termination process is assisted by special proteins called termination factors or release factors (RF1, RF2, and RF3) which recognize the stop codons. Their association (their binding to the A site) stimulates the release of the polypeptide from the trna in the P site. The trna is then released. The ribosome divides into the large and small subunits, and become ready for synthesizing another polypeptide. 16
Amino terminal end Carboxyl terminal end 17