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‘ase protein, 1s then trimmed to the mature, the polyprotein requires a translational, he UAG termination codon at the end of, units of £. coli, DNA polymerase 11, , used for the gag protein alone. The polyprotein, or gag-polymer, reverse transcriptase by proteolytic digestion. Production of, frameshift in the overlap region to allow the ribosome to bypass t, the gag gene. A similar mechanism produces both the t and y sub, from a single dnaX gene transcript., , ‘CODON-tRNA INTERACTIONS: WOBBLE HYPOTHESIS oe, , When several different codons specify one amino acid, the difference between them usually lies at the, , ‘third base position (at the 3” end), For example, alanine is coded by the pies GCU, GCC, GCA, ang, d :, , GCG. The codons for most amino acids can be symbolized by XY ¢ or XY. The first two letters of each, , codon are the primary determinants of specificity. Transfer RNAs base-pair with mRNA codons at a, three-base sequence on the tRNA called the anticodon. The first base of the codon in mRNA (read in the, $‘-43’ direction) pairs with the third base of the anticodon therefore a specific tRNA anticodon would, exist for every codon. If the anticodon triplet of a tRNA recognize only one codon triplet through, Watson-Crick base pairing at all three positions, cells would have a different tRNA for each amino acid, codon, therefore at least 61 different tRNAs, possibly with an additional 3 for the chain-terminating, codons, would be present. This is not the case, however because the anticodons in some tRNAs include, the nucleotide inosinate (designated I; Adenine derivative; contains the uncommon nitrogenous base, hypoxanthine). Inosinate can form hydrogen bonds with three different nucleotides (U, C, and A), although these pairings are much weaker than the hydrogen bonds of Watson-Crick base pairs, (G=C; A=T)., , tRNA, Anticodon, mRNAS' a, 321 3241 324, auilosten ae | Fe} Tel 6), & ' i, Condon (5")C-G-A C-G-U 6-6-6 (3°, 123 125 § 3 G6 ), , In 1966, Francis Crick devised the wobble hypothesis to explain these observations, Examination of, these and other codon-anticodon pairings led Crick to conclude that the third base of most codons pairs, rather loosely or “wobbles” with the corresponding base of its anticodon. This hypothesis predicted, existence of atleast two tRNA for each amino-acid wih codons that exhibit complete degeneracy., , posed a set of four relationships called the wobble hypothesis:, 1. The first two bases of an mRNA codon always form strong Watson:, corresponding bases of the tRNA anticodon and confer most of the codin;, , , , , Crick base pairs with the, ig specificity., , The first base of the anticodon (that pairs with the third base of the codon) determines the number of, codons recognized by the tRNA. When the first base of the anticodon is C or A, base pairing is

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Molecular Biology 249, specific and only one codon is recognized by that tRN, ss specific and two different codons may be read. W, ess §, ofan anticodon, three different codons can be Trecogniz, , A. When the first base is U or G, binding is, hen inosine (1) is the first (wobble) nucleotide, , baa ed-the maximum number for any tRNA., When an amino acid is specified by several different, , a ae odons, the codons that differ in either of the, first two bases require different tRNAs., , , Aminimum of 32 tRNAs are required to translate all 61 codons (31 to encode the amino acids and 1, for initiation)., , 4. The wobble (or third) base of the codon contributes to specificity, but, because it pairs only loosely, with its corresponding base in the anticodon, it Permits rapid dissociation of the tRNA from its codon, , during protein synthesis. If all three bases of a codon engaged in strong Watson-Crick pairing with, the three bases of the anticodon, tRNAs would dissociate too slowly and this would severely limit the, , rate of protein synthesis. Codon-anticodon interactions balance the requirements for accuracy and, speed., , , , 7 5 5, Anticodons in, anticodon iticodon, Pn onaNA aA, iil iti, mRNA 5° x 5 ey ee, 123,, Codon Codon, Cin the first (5°) anticodon Ain the first (5°) anticodon, , position can only pair with G (3°) in codon position can only pair with U (3°) In codon, , Recognition of Two Codons|, , , , 3 * ge oe ah gs, Anticodons in ‘Anticodons in Anticodons, anticodon arm anticodon arm ‘anticodon arm amucodonarh, JY of tRNA Ye of tRNA J? oftRNA YY of tRNA, 324 324 324 321, i iii, Say — mRNA 5"| ave a to +, 123, 23,, Unte Codon Codon Coden Codon, (5) anscodon position can pair with, , elther G or A (3’) In codon G in the first (5°) anticodon position can pair with either C of U (3°) in codon, , , , , , , , , , , , , , , , , , , , , , , , , , 3 5 a 5 5, iticodon: Anticodons in Anticodons in, anne mn anticodon arm ‘|anticodon arm, i of tRNA 17 of tRNA 17 ‘of tRNA, RNA 5’ MEX 3S 5 XY CS 3° 5 EX’. s, 123, 123, 123,, Codon Codon Codon, , Jin the first (6") anticodon position can palr with either A, C or U (3°) In codon, é Wobble base of the anticodon determines the number of codons recognized by tRNA

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Translation is the process of translating the sequence of a messenger RNA (mRNA) molecule to a, , sequence 0 of amino acids during protein synthesis, ‘The machinery responsible for translation of mRNAs_, into o proteins is composed of four primary components:. 7 , 1, Messenger RNA (mRNA),, 2, Transfer RNA (tRNA),, , 3, Ribosome and, , , , 4, Aminoacyl-tRNA synthetases, , MESSENGER RNA (mRNA), , The mRNA provides the information that must be interpreted by the translation machinery and is the, ae for translation. The protein- coding region of the mRNA consists of an ordered series of three, , nucleotide-long units called codons that specify the order of amino acids. The tRNAs provide the,, as face between the amino acids is being added to the growing polypeptide chain and the codons, : oe mRNA. Enzymes called aminoacyl-tRNA synthetases couple an amino acids to specific tRNAs that., Tecognize | ee > appropriate codon(s). The final major player in translation is the ribosome, a remarkable,, , multi-mega-dalton machine composed of both RNA and protein. The ribosome coordin: nates the correct, recognition of the mRNA by each tRNA and catalyzes peptide-bond f formation between the growing, ‘polypeptide chain and the amino acid attached to the he selected (RNA., , The protein coding region of each mRNA is composed of a contiguous, non- -overlapping string of, codons called open reading frame (ORF). Each ORF specifies a single protein and starts and ends at the, intemal sites within the mRNA. ‘Translat nslation starts at ts at the 5’ end of the ORF and proceeds one codon at a, , he 3’ end. The first and last codons of an ORF are known as the start and stop codons., , , , , , The start codon has two important functions4) Itspecifies the first amino acid to be incorporated in the growing polypeptide chain., b) w that any stretch of MRNA could, , It defines the reading frame for all subsequent codon. As we kno, ¢ location of first codon, the start, , be translated in three different reading frames. Thus, by setting th, Codon determines the location of all following codons., , ao Okaryotes, start codon is usually 5’AUG 3’, but 5’GUG 3” and som, , = eukaryotes, 5’AUG 3’ always acts as s tart codon., , netimes even 5’UUG 3’ are”

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are erase, called GSaemilinpensy Ee ina template independent manner. On the other hand, the, eg oF RNA terminates wih the S or ‘C’ residue. A second striking aspect of tRNAs is the presence, 5 | unusual bases In their primary structure that are added post-transcriptionally by enzymatic, , sever@ : : :, ion of normal bases in the polynucleotide chain. For example, pseudouridine (y) dihydrouridine, , sficat, odifica is i: . r, a sonisinic acid, methyl cytosine, methylguanine, ribothymidine etc., ( Is, , The primary structure is first sequenced by Holley er al. 1965 for yeast alanine tRNA. The tRNA, gloverteat secondary structure consist of a series of loops separated by short stems of helical double, giranded regions of four to seven complementary bases long. The loops are:+, , Molecular Biology 253, , The acceptor end (5° CCA 3’): The 5° CCA 3’ sequence at the extreme 3’ end of the molecule is a, single-strand region that protrudes from the double-strand stem. It is the site of attachment of the, specific amino acid., , The T loop or TwC loop: It has the conserved sequence 5’-TyCG-3’. It is ribosome recognition site, and interacts with a complementary region of 5S rRNA during protein synthesis., , + The D loop: It contains the characteristic dihydrouridines in the loop., , + The anticodon loop: It contains the anticodon, a three-nucleotide-long sequence that is responsible, for recognizing the codon by base pairing with the mRNA codon. The anticodon is always bracketed, on the 3’ end by a purine and on its 5’ end by uracil., , «The variable loops: It lies between the anticodon loop and the TyC loop. It is highly variable in size, and varies in size from 3 to 21 bases., , ‘Precise aminoacylation of tRNA by its cognate aminoacyl-tRNA synthetases, depend on enzyme, interaction with the acceptor stem « and the anticodon loop. The acceptor stem is an especially important, determinant for the specificity of tRNA synthetase recognition. In some cases, changing a single base in, the acceptor stem (known as the discriminator base) is sufficient to convert the recognition specificity of, a tRNA from one synthetase, to another. The anticodon., loop frequently contributes, 0 discrimination as well., , wU loop Discriminator base, , , , ‘Acceptor end, wl loop Acceptor arm Acceptor stem, , X-ray crystallography, reveals an L-shaped tertiary, (3D) structure of tRNA in, which the terminus of the, acceptor stem is at one end, of the molecule and the, anticodon loop is ~70A, away at the other end. In, L-shaped conformation, one, leg of the L is formed by the, acceptor and T loop folded into a continuous A-RNA-like double helix and the other leg is similarly, composed of the D and anticodon stems. The L-shaped structure is stabilized by different types of, structure like- base-stacking interactions as found in double-stranded DNA resulted from base pairing;, hydrogen bonds between bases in different helical regions resulted from unconventional (non-WatsonCrick) bonding; and interactions between the bases and the sugar-phosphate backbone., , , , Anticodon, , 3D structure of tRNA