BIOC201 - Nucleic Acid, DNA, Protein Synthesis Part 2 PDF

5. STUCTURE OF RNA - RNA, like DNA, is a long unbranched macromolecule consisting of nucleotides joined by 3’o5’ phosphodiester bonds. - The number of nucleotides in RNA range from as few as seventy five to many thousands. - The molecular structure of RNA differs from DNA in two ways...

5. STUCTURE OF RNA - RNA, like DNA, is a long unbranched macromolecule consisting of nucleotides joined by 3’o5’ phosphodiester bonds. - The number of nucleotides in RNA range from as few as seventy five to many thousands. - The molecular structure of RNA differs from DNA in two ways: i) The sugar unit in RNA is D-Ribose rather than the 2-deoxy-D-ribose that is present in DNA. Ribose contains a 2’-hydroxyl group that is not present in deoxyribose. ii) The nitrogenous base Uracil is found in RNA instead of thymine which is present in DNA. Uracil like Thymine can only base pair with Adenine. H3 C Thymine (DNA) Uracil (RNA) 6  NH2 N N Adenine O- N N O P O NH2 O O N Cytosine O OH O P O N O O O- O N NH Guanine O OH N N NH2 O P O O O O- NH O OH Uracil O P O N O O- O O OH Figure 7: RNA strand structure In a RNA strand the ribonucleotide residues are connected to each other by 3'-5’- phosphodiester linkages. 6. Types of RNA - Several types of RNA participate in the transfer of information from DNA to protein. - tRNA binds and carries amino acids to the ribosome during protein synthesis - rRNA, (together with several proteins) constitute the ribosome. - mRNA specifies the amino acid sequence during protein synthesis. - Lastly, small RNA molecules, usually having catalytic activity, are involved in processing of the above RNA molecules. - In E. coli, the cell is able to devote up to 60% of its RNA synthesizing ability for the production of mRNA, yet, mRNA only accounts for about 3% of the total cellular RNA. This is because of varying stabilities between the RNA species; tRNA and rRNA are very stable, whilst mRNA is rapidly degraded after translation. Why is mRNA generally more unstable than tRNA and rRNA? 7 7. Translation (Protein Synthesis) - The essential features of translation were mapped out between 1950 – 1965 due to investigations by Paul Zamecnik, Mahlon Hoagland and Francis Crick. 7.1 The Genetic Code ƒ The genetic code (Figure 8) refers to the triplet base sequences in mRNA (or DNA) that specify the amino acids to be incorporated into a protein. This triplet base sequence is called a codon. ƒ The genetic code contains all the information for initiating translation, specifying amino acid sequence in a protein’s primary structure, terminating translation and releasing the nascent protein. ƒ The genetic code is nonoverlapping and made up of 64 codons. eg. AUGCCA……… ƒ Codon AUG signals initiation of translation and encodes methionine. In some organisms, GUG serves as the start signal and encodes valine. For this reason, many proteins begin with met or val. (Note: in prokaryotes, only the first AUG at the 5’ end encodes a modified methionine residue called formyl-methionine; all other AUG codons encode methionine). ƒ Termination of translation is signalled by termination (stop or nonsense) codons UAA, UAG and UGA (sometimes referred to by the classical terms, amber, ochre and opal, respectively). These codons do not encode amino acids. ƒ The remaining 61 codons specify the 20 standard amino acids. Different codons can encode the same amino acid (synonym codons), e.g. aspartate, cysteine and histidine each have two different codons; alanine and proline each have four different codons and leucine and serine each have six different codons. The genetic code is therefore regarded as being degenerate in that many AA’s are designated by more than one triplet. However, no codon specifies more than one amino acid, i.e. the genetic code is also unambiguous. 8  Figure 8. The standard genetic code. 7.2 Transfer RNA ƒ Transfer RNA (adaptor) molecules interpret the genetic code and are the crucial link between the genetic code in mRNA, and the sequence of amino acids in a protein. ƒ The secondary structure of tRNA resembles a clover-leaf (Figure 9). This pattern arises due to loop and stem formations. Stem formations arise due to self- complementary intrachain base pairing. Most tRNA molecules are about 73 – 93 nt long. ƒ The amino acid arm (at 3’ end) serves as the point for AA attachment (esterification) and subsequent transport to the ribosome. ƒ The anticodon loop contains the anticodon which forms a complementary base pair interaction with the codon in mRNA. ƒ The DHU and T\C arms may play a role in binding of the tRNA to the ribosome. A variable arm may be present / absent. ƒ A feature of tRNA is that it contains unusual bases (7-15 per tRNA) that include: 9  a) unique bases (e.g. dihydrouridine, pseudouridine) b) covalently modified bases (e.g. methyl-G and methyl-C). These bases may assist the tRNA to spontaneously fold into its native conformation and also protect against nuclease attack. ƒ For tRNA to fulfill its role a cell must contain at least 20 tRNA species (one for each amino acid) and each tRNA must be capable of at least recognising one of the 61 codons encoding amino acids. \ = pseudouracil dihydrouridine Figure 9. Structure of tRNA. 10 7.3 Aminoacyl–tRNA synthetases - When a specific amino acid is esterified to the 3’ end of a correct tRNA molecule, an aminoacyl-tRNA is formed. This reaction is catalyzed by aminoacyl-tRNA synthetases. - Aminoacyl-tRNA is a high-energy molecule; the attached amino acid is therefore “activated” for incorporation into a polypeptide. Amino acids on their own cannot recognize codons. - A specific aminoacyl-tRNA molecule is identified by naming both the tRNA and amino acid it is attached to, e.g. alanyl-tRNAala. - It is possible for an amino acid to be esterified to the incorrect tRNA, e.g. valyl- tRNAleu. However, aminoacyl–tRNA synthetases have an error checking function and rapidly correct such errors. - The overall reaction for amino acid activation is: amino acid + tRNA + ATP o aminoacyl-tRNA + AMP + PPi. 7.4 Translation in Prokaryotes (E. coli) The translation process contains three distinct phases, viz. initiation, elongation and termination. These phases require mRNA to be translated, 30S and 50S ribosome subunits, aminoacyl-tRNA molecules and accessory proteins (initiation, elongation and release / termination factors). 7.4.1 Initiation (Figure 10) - Initiation factors 1 and 3 bind to the 30S ribosome subunit. IF-3 prevents the 30S subunit from prematurely associating with the 50S subunit. IF-1 is thought to promote activity of the IF-2 and IF-3. - A complex of [IF-2–GTP] complex binds to formylmethionine-tRNAfmet. The resulting [IF-2–GTP-formylmethionine-tRNAfmet] complex binds to the 30S subunit. tRNAfmet is called the initiator tRNA molecule and IF-2 is responsible for recognizing and transporting this tRNA to the 30S subunit. - The 30S subunit then binds to mRNA. This binding is specific and results in the initiation codon (AUG) being positioned in the P (peptidyl) site. The binding specificity is mediated by the Shine-Dalgarno sequence (near the 5’ end in mRNA) forming a complementary base pair interaction near the 3’ end of 16S RNA in the 30S subunit. IF-3 may assist with the specificity of these interactions. - Within the P site, a codon-anticodon interaction forms between the AUG initiation codon and the anticodon of formylmethionine-tRNAfmet. 11 - The GTP in IF-2–GTP is hydrolyzed releasing IF-2–GDP and Pi. This hydrolysis also releases IF-1 and IF-3. - The 50S subunit then joins the [mRNA–formylmethionine-tRNAfmet] complex to collectively form the 70S initiation complex. Figure 10. Initiation of prokaryotic translation. 12 13 7.4.2 Elongation 7.4.3 Termination 7.5 Regulation of Translation - Translational regulation is essential in controlling gene expression and generally provides a means of controlling cellular protein levels. - Regulation of translation can be achieved at during the initiation, elongation or termination phases of protein synthesis. - In prokaryotes, the synthesis of proteins that constitute ribosomes are coupled with the rate at which ribosomes are assembled (Note: ribosome = proteins + rRNA). As long as ribosomal proteins are incorporated into ribosomes, the mRNA molecules encoding these proteins are translated. When ribosome assembly slows down, the excess ribosomal proteins bind to their own mRNA and further translation is blocked. 14