PTT 207 Biomolecular and Genetic Engineering Semester 2 2013/2014 BY: PUAN NURUL AIN HARMIZA ABDULLAH
Mechanism of Translation 1. Initiation 2. Elongation 3. Termination
Initiation of Translation • Initiation of translation in prokaryotes involves the assembly of the components of the translation system which are: 1. the two ribosomal subunits (50S & 30S subunits), 2. the mRNA to be translated, 3. the first (formyl) aminoacyl tRNA (the tRNA charged with the first amino acid), 4. GTP (as a source of energy), and 5. three initiation factors (IF1, IF2, and IF3) which help the assembly of the initiation complex.
1. Ribosome Structure and Assembly Structure of Ribosome • The bacterial 70S ribosome is composed of: • Large 50S subunit = assembled from a 23S rRNA and a 5S rRNA • Small 30S subunit = assembled from a single 16S rRNA. • S = Svedberg unit • Functions: • Decoding the genetic code in mRNA • Catalyzing the formation of peptide bonds between amino acids resulting a polypeptide.
• There are 3 tRNA-binding sites on the ribosome: • A (acceptor) - entry point of aminoacyl-tRNA (except first aminoacyl-tRNA which enters at P site. • P (peptidyl) - where the peptidyl tRNA is formed in the ribosome. • E (exit) - exit site of the now uncharged tRNA after it gives its amino acid to the growing peptide chain.
2. mRNA • mRNA is the messenger RNA. • It's the only type of RNA that carries protein-building information. • the mRNA carries the codons i.e the A,U,G, and C codons. What about tRNA and rRNA?....
2. mRNA The Genetic Code • A group of three adjacent nucleotides in DNA is transcribed into three complementary RNA nucleotides, which in turn are translated into a single amino acid within a polypeptide chain. • Being a triplet code, 4x4x4 = 64 different combinations exist, which is many more than are necessary to encode 20 different amino acids. • Each coding triplet is referred to as a codon. • Each mRNA codon in next Table is conventionally written with the 5' nucleotide at the left and the 3' nucleotide at the right, because protein synthesis begins at the 5' ends of mRNA molecules and proceeds toward their 3' ends.
Table : Codons (displayed as mRNA triplets)
Figure 4.5 pg 69
2. mRNA The Genetic Code • The code is highly degenerate in that more than one codon can specify the same amino acid. • Because of code degeneracy, many changes (mutations) can occur in a gene that will have no effect on the amino acid composition of the gene product. • Such changes are referred to as silent mutations.
2. mRNA The Genetic Code • The complementary base pairing between an mRNA codon and its anticodon in a tRNA is usually much less restrained at the third position than in the other two positions of the triplet. This phenomenon, called wobble, allows the same tRNA to recognize more than one mRNA codon in many cases. • Sixty-one codons are sense codons that specify amino acids. • There are three codons that are not recognized by any tRNA: UAA, UAG, and UGA. • These are termed nonsense codons or stop codons, because they provide part of the signal that protein synthesis should stop at that point.
2. mRNA The Genetic Code • Translation in prokaryotes begins at a start codon. The bacterial mRNA translation initation codon (AUG) encodes N- formylmethionine, whereas internal AUG codons specify methionine. • The 3'-UAC-5' anticodon of the tRNA pairs (in antiparallel fashion) with the complementary 5'-AUG-3' codon in the mRNA.
3. Special Initiator tRNA • The initiator tRNA is called fMet-tRNA. It differs from all other tRNAs by one less base pair in the acceptor stem region and by three consecutive G:C base pairs in the anticodon region . These changes alter the 3-dimensional structure of the fMet-tRNA in subtle ways. • fMet-tRNA fMet and Met-tRNA Met both recognize the AUG codon. • A methionine is attached to both tRNAs by the same methionyl tRNA synthetase. However, once the methionine is attached to either tRNA, a special enzyme called formyl transferase, which recognizes only the initiator tRNA fMet adds a formyl group to the amide nitrogen. Because the amide N is now blocked from further reaction, this amino acid can only be positioned at the start of a polypeptide chain.
Only one special tRNA is used to place the first amino acid, which is always a formyl-methionine (fMet):
4. Guanosine triphosphate • Guanosine-5'-triphosphate (GTP) is a purine nucleoside triphosphate. • GTP is used as an energy source for the binding of a new amino-bound tRNA to the A site of the ribosome. GTP is also used as an energy source for the translocation of the ribosome towards the 3' end of the mRNA.
5. IF1, IF2, and IF3 • Prokaryotes require the use of three initiation factors: IF1, IF2, and IF3, for translation. • The initiation factors are proteins that facilitate the assembly of an active ribosomal particle and the placement of the first fMet-tRNA fMet . • IF3 binds to 30S subunit and promotes the dissociation of the 30S subunit from the 50S subunit and the subsequent binding of the mRNA. IF1 helps IF3 by increasing the dissociation rate between the 30S and 50S subunit. • IF2 , in complex with GTP, binds to fMet-tRNA fMet and places it in the P-site of the 30S subunit. Then IF3 is released, causing GTP to hydrolyze to GDP, which in turn releases IF2-GDP. GTP hydrolysis promotes the release of IF1 and the subsequent association of the 30S subunit with the 50S subunit.
Shine-Dalgarno Sequence • To distinguish the start AUG codon from AUG codons which code for internal methionines or from a fortuitous AUG combination in another reading frame, the start AUG codon in prokaryotes is preceded by a highly conserved sequence at the 5´ end of the mRNA transcript which serves as a ribosome-binding site. The Shine- Dalgarno sequence is a purine-rich tract of 3-10 nucleotides and precedes the start AUG codon by approximately 10 nucleotides upstream on the 5´ side. The Shine-Dalgarno sequence forms base pairing with a highly conserved, pyrimidine-rich region of the 16S rRNA which is part of the 30S ribosomal subunit. This base-pairing properly aligns the start AUG codon in the P site of the 30S ribosomal subunit. In eukaryotes, protein synthesis usually begins with the first AUG codon from the 5´ end. Eukaryotic mRNA has a special cap at the 5´ end of the mRNA which is recognized by a cap- binding protein.
Aminoacyl-tRNA Synthetases Aminoacyl-tRNA Charging • All aminoacyl-tNRA synthetases attach an amino acid to a tRNA in 2 enzymatic steps: 1. The AA reacts with ATP to become adenylylated by the addition of AMP, and pyrophosphate is released. The AA attached by a high-energy ester bond between the carbonyl group of AA and the phosphoryl group of AMP. 2. AMP is released and the AA transferred to 3’ end of tRNA via the 2’OH for class I enzymes and via the 3’OH for class II enzymes. What are class I and II enzymes?
Figure 14.6 pg 456
TRANSCRIPTION MECHANISM
Initiation Mechanism • In the first step of translataion initiation, binding of IF1 & IF3 to an empty 70S ribosome dissociates it into 50S and 30S subunits. • 5' region of mRNA (Shine-Dalgarno (S-D) recognition sequence) binds to 16S rRNA of free 30S ribsomal subunit, IF3 is released. Initiator fMet-tRNA fMet carried by IF2-GTP binds to P site of 30S ribsomal subunit. IF3 is released. • And then GTP hydrolysis releases IF1, IF2, GDP, and phosphate, allowing the 50S subunit to join the 30SmRNA- tRNA fMet complex to form a complete 70S initiation complex. The tRNA fMet ends up in the P site of the ribosome. This completes the initiation phase.
Formation of the 70S initiation complex 1. Binding of IF1 & IF3 to an empty 70S ribosome dissociates it into 50S and 30S subunits. • 5' region of mRNA binds to 16S rRNA of free 30S ribsomal subunit, IF3 is released. 2. Initiator fMet- tRNA fMet carried by IF2-GTP binds to P site of 30S ribsomal subunit. 3. Free 50S ribosomal subunit binds, IF1 & IF2-GDP + P i are released.
Elongation Mechanism • Elongation of the polypeptide chain involves addition of amino acids to the carboxyl end of the growing chain. • The growing protein exits the ribosome through the polypeptide exit tunnel in the large subunit. • Starts when the fMet-tRNA enters the P site, causing a conformational change which opens the A site for the new aminoacyl-tRNA to bind. This binding is facilitated by elongation factor,Tu (EF-Tu). • Now the P site contains the beginning of the peptide chain of the protein to be encoded and the A site has the next amino acid to be added to the peptide chain. The growing polypeptide connected to the tRNA in the P site is detached from the tRNA in the P site and a peptide bond is formed between the last amino acids of the polypeptide and the amino acid still attached to the tRNA in the A site. This process, known as peptide bond formation , is catalyzed by a ribozyme (the 23S ribosomal RNA in the 50S ribosomal subunit).
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