NUCLEIC ACIDS AND PROTEIN SYNTHESIS ǁ Author: DONGO SHEMA F. ǁ JULY 2016 ǁ

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1 NUCLEIC ACIDS Examples: 1. Deoxyribonucleic acid (DNA) 2. Ribonucleic acid (RNA) GENERAL STRUCTURE OF NUCLEIC ACIDS (Describe the structure of nucleic acids /a nucleic acid) Nucleic acids are polymers made of subunits called nucleotides. A nucleotide is made up of three molecules: (i) Phosphate group (ii) Pentose sugar either Deoxyribose (in DNA) or Ribose (in RNA) (iii) Nitrogen base any purine (Adenine, Guanine) or pyrimidine (Cytosine and either Thymine in DNA or Uracil in RNA) Nucleoside forms when a pentose sugar joins an organic base by condensation reaction (a water molecule is lost). Nucleotide forms when a nucleoside (pentose sugar + organic base) joins a phosphate by loss of second water molecule. The sugar-phosphate-sugar backbone is formed when the 3 carbon on one sugar joins to the 5 carbon on the next sugar by phosphodiester bonds repeatedly to form a polynucleotide (long chain of nucleotides) with organic bases protruding sideways from sugars. Nitrogen base Phosphodiester bonds Pentose sugar COMPONENTS OF NUCLEOTIDES MOLECULE DNA RNA Pentose sugar Phosphoric acid Purines (Double ringed organic bases) Pyrimidines (Single ringed organic bases) Page 1 of 8

2 DNA STRUCTURE ACCORDING TO WATSON AND CRICK DNA is a very large polymer of nucleotides. A DNA nucleotide is made up of three molecules: (i) Phosphate group (ii) Deoxyribose sugar (iii) Nitrogen base - either adenine (A), guanine (G), thymine (T) or cytosine (C). The polynucleotide stands are antiparallel (face in opposite directions) i.e. one runs from 3 to 5 direction while another runs from 5 to 3 direction. Untwisted DNA is ladder-like, in which the sugarphosphate backbones represent the handrails while the nitrogen base pairs represent the rungs. Twisted DNA forms a double helix of major and minor grooves. The sugar-phosphate-sugar backbone is held by covalent phosphodiester bonds, while the nitrogen bases from the two strands form weak hydrogen bonds by complimentary base pairing i.e. A with T, C with G. ADAPTATIONS OF DNA (i) Sugar-phosphate backbone is held together by strong covalent phosphodiester bonds to provide stability. (ii) The two sugar-phosphate backbones are antiparallel which enables purine and pyrimidine nitrogen bases to project towards each other for complimentary pairing. (iii) Sugar-phosphate backbones are two / it is double stranded to provide stability. (iv) The two sugar-phosphate backbones form a double helix to protect bases/hydrogen bonds. (v) Long/large molecule for storage of much information. (vi) Double helical structure makes the molecule compact to fit in the nucleus. (vii) Base sequence allows information to be stored. (viii) Double stranded for replication to occur semiconservatively/ strands can act as templates. (ix) There is complementary base pairing / A-T and G-C for accurate replication/identical copies can be made; (x) Weak hydrogen bonds enable unzipping/separation of strands to occur readily. (xi) There are many hydrogen bonds which increase stability of DNA molecule. Sugar-phosphate backbone Base pair Minor groove Major groove 20 Å 34 Å Hydrogen bonds THEORIES OF DNA REPLICATION DNA replication is the process by which the parent DNA molecule makes another copy of itself. 1. Fragmentation hypothesis (Dispersive hypothesis) The parent DNA molecule breaks into segments and new nucleotides fill in the gaps precisely. 2. Conservative hypothesis The complete parent DNA molecule acts as a template for the new daughter molecule, which is assembled from new nucleotides. The parent molecule is unchanged. 3. Semi-conservative hypothesis The parent DNA molecule separates into its two component strands, each of which acts as a template for the formation of a new complementary strand. The two daughter molecules therefore contain half the parent DNA and half new DNA. The semi conservative hypothesis was shown to be the true mechanism by the work of Meselsohn and Stahl (1958) in their experiment on bacterium E.coli using radioactive 15 N. Page 2 of 8

3 Illustration of three possible theories of DNA replication Parent DNA Old strand New strand Dispersive Conservative Semi-conservative DESCRIPTION OF DNA REPLICATION DNA replication is the process by which parent DNA molecule makes another copy of itself, semi conservatively (1 new, 1 old strand together). It occurs during the synthesis phase of interphase. DNA Helicase enzyme untwists and unzips DNA by breaking the hydrogen bonds to expose the bases, creating the Y-shaped replication fork, the two opened strands of DNA behind it (DNA is replicated a bit at a time and the whole molecule is never completely uncoiled). RNA primase enzyme lays down an RNA primer at the 3 end of the old DNA strand to guide the action of DNA polymerase. DNA polymerase enzyme removes the RNA primer from the new strand then moves along the exposed base sequences, attaching free DNA nucleotides of complementary bases to create a new DNA strand as it goes. DNA ligase joins adjacent Okazaki fragments on the lagging strand (new strand laid down in the opposite direction of the replication fork) and any sections of new DNA on the leading strand (new strand laid down in the direction of the replication fork) that need to be joined. DNA polymerase reads the exposed code from 3 to 5 end and therefore assembles the new strand from 5 to 3 end Several molecules of DNA polymerase act simultaneously at multiple sites, each assembling a separate section of the new strand of DNA. These new DNA segments are then joined together by the enzyme DNA ligase. The two new daughter molecules then coil up again to reform the double helix structure. Illustration of DNA replication Page 3 of 8

4 GENERAL STRUCTURE OF RNA RNA molecules are small/short, single stranded (rrna and mrna) or double stranded (trna) polymer of nucleotides. RNA nucleotide is made up of three molecules: (i) Phosphate group (ii) Ribose sugar (iii) Nitrogen base - either adenine (A), guanine (G), cytosine (C) or uracil (U) The sugar-phosphate-sugar backbone is held by covalent phosphodiester bonds. RNA occurs in three types whose sizes, shapes, amounts/abundance and roles vary: 1. Ribosomal RNA (rrna) Forms 80% of the total RNA in a cell. rrna in different species vary in size e.g. in humans 18S rrna has 1868 nucleotides while 28S rrna has Permanently combined with protein to form catalytic component of ribosomes. Manufactured in nucleolus. rrna is a site of protein synthesis in cells. 2. Messenger RNA (mrna) Forms 3-5% of the total RNA in a cell. Single stranded polynucleotide chains with 5 to 3 polarity Average size of eukaryotic mrnas is 1500 to 2000 nucleotides Manufactured in the nucleus mrna carries coded information from DNA to ribosomes in the cytoplasm 3. Transfer RNA (trna) Forms about 15% of the total cell RNA. Primary structure in all trnas has sequences of 73 to 93 nucleotides. 3 end always terminates with the sequence CCA, where amino acid attaches while the 5 end terminates in base G. Secondary structure forms a clover leaf shape with 4 hydrogen bonded base-paired stems Cloverleaf contains three non-base-paired loops: (i) Amino acyl synthetase binding loop (ii) Anticodon (iii) Ribosomal binding loop. Tertiary structure is a compact L shape whereby the anticodon stem and acceptor stem form a double helix. Anticodon is a single stranded loop at the bottom. trna carries amino acids in the cytoplasm to ribosomes. Adaptations of trna: (1) Active sites (anticodon and amino acid) are maximally separated to avoid interference (2) Small size for mobility readily. COMPARISON OF DNA AND RNA Similarities Both: (1) are polymers of nucleotides (2) carry genetic information (3) have same purine bases adenine and guanine plus pyrimidine bases cytosine (4) originate from the nucleus (5) occur in the cytoplasm Differences Aspect Deoxyribonucleic Acid (DNA) Ribonucleic Acid (RNA) (i) It s the blueprint of biological guidelines in (i) Helps carry out DNA s blueprint guidelines. Function organisms (ii) Stores genetic information for a long time and (ii) Transfers genetic code needed for the creation transmits it. of proteins from the nucleus to the ribosome. Structure (iii) Double-stranded. (iv) Hydrogen bonds occur between complementary nitrogen bases of opposite strands (A-T, C-G) (v) Spirally twisted to produce a regular helix (vi) Occurs in form of chromatin or chromosomes (iii) Single-stranded. (iv) Base pairing through hydrogen bonds occurs in the coiled parts (v) The strand may fold at places to form a secondary helix (vi) Occurs in ribosomes or forms association with ribosomes Page 4 of 8

5 Base Pairing Location Stability Propagation Unique Features Size Types (vii) Adenine links to thymine (A-T) (viii) Purine and pyrimidine bases are in equal number (ix) Much of DNA is in the nucleus of a cell, little in mitochondria and chloroplasts. (x) Deoxyribose sugar in DNA is less reactive because of C-H bonds. (xi) Stable in alkaline conditions. (xii) Long lived (xiii) DNA is self-replicating. (xiv) DNA is protected in the nucleus, as it is tightly packed. (xv) Very large/long (has over a million nucleotides) (xvi) Quantity is fixed in a cell (xvii) Only two types: intra nuclear and extra nuclear DNA (vii) Adenine links to uracil (A-U) (viii) No proportionality between numbers of purine and pyrimidine bases. (ix) Much of RNA is in the cytoplasm, little in the nucleus. (x) Ribose sugar is more reactive because of C- OH (hydroxyl) bonds. (xi) Not stable in alkaline conditions. (xii) Some RNA are very short lived while others have somewhat longer life. (xiii) RNA is synthesized from DNA when needed. (xiv) RNA strands are continually made, broken down and reused. (xv) Much shorter (Depending on the type, RNA contains 70 12,000 nucleotides). (xvi) Quantity is variable (xviii) Three different types: mrna, trna and rrna THE CENTRAL DOGMA OF MOLECULAR BIOLOGY It states that DNA makes RNA makes proteins PROTEIN SYNTHESIS Protein synthesis is the process by which individual cells construct proteins. Protein synthesis occurs in separate but interrelated steps as follows: 1. TRANSCRIPTION Transcription is the process whereby the DNA code of a gene is copied to make messenger RNA (m-rna). Importance of transcription (i) DNA is too large to fit through the nuclear pores, yet mrna being small can readily exit the nucleus. (ii) DNA contains many codes that aren t always needed at a given time, so m-rna only carries that code needed to make specific proteins out of the nucleus to the ribosome. Transcription is performed by an enzyme called RNA polymerase and a number of accessory proteins called transcription factors which together form the transcription initiation complex. Transcription is initiated when RNA polymerase enzyme: (i) Binds to the DNA strand at a promoter. The promoter is the beginning code of DNA on the gene where the RNA polymerase begins reading the DNA code, and is always the 3 letter triplet TAC. (ii) Untwists then unzips the two strands of DNA. (iii) Reads the antisense strand of DNA from 3 to 5 (antisense - side containing the code to make a polypeptide). (iv) Matches new nucleotides with their complements on the DNA strand (G with C, A with U) in the 5 to 3 direction. (v) Binds these new RNA nucleotides together to form a complimentary copy of the DNA strand (mrna) The sense strand of DNA is the side of DNA that does not contain a code to make a polypeptide. It is not read by the RNA polymerase. Elongation occurs when the new m-rna strand is lengthened in the 5-3 direction. Transcription stops when the terminator code is section of DNA is reached. Its code is complementary to the stop codons of m-rna. The newly created m-rna of eukaryotic cells is edited by removal of non-coding sections, called introns since they remain in the nucleus. The finished m-rna (with exons only) containing the code needed to make the polypeptide leaves via nuclear pores to the ribosome in the cytoplasm for translation. Page 5 of 8

6 2. TRANSLATION Translation involves three main steps: (1) Initiation (2) Elongation (3) Termination Initiation occurs when the small ribosomal subunit binds at 5 end of mrna molecule where there is the start codon AUG. trna molecule carrying the amino acid methionine binds to the ribosomal small sub-unit corresponding to the start codon (AUG) on mrna. The large ribosomal subunit binds on the small sub-unit to complete the initiation complex. The large ribosomal subunit has two amino acid binding sites: peptidyl site (P-site) and amino acyl site (A-site). Elongation occurs when the first trna with anti-codon UAC (carrying the amino acid methionine) fits into the P-site of the ribosome while the second trna, whose anti-codon compliments the codon on mrna fits into the A-site of the ribosome. A peptide bond forms between the methionine and the amino acid carried by the second trna. The methionine-specific trna leaves the P-site while the ribosome shifts so that the second trna now occupies the P-site, allowing the third codon on mrna to occupy the A-site. A third trna (carrying the third amino acid), whose anti-codon compliments the codon on mrna fits into the A-site of the ribosome, enabling the formation of a second peptide bond between the second and third amino acids. The second trna leaves the P-site while the ribosome shifts down one codon so that the third trna now occupies the P-site, allowing the fourth codon on mrna to occupy the A-site. Termination occurs when all the codons on mrna are read. The ribosome reaches one or more stop codons on mrna (UAA, UAG, UGA). The ribosome detaches from the mrna and splits into its small and large sub-units, while the new protein floats away Note: 1. Several ribosomes can attach to a molecule of mrna (to form polysomes/polyribosomes) one after another so that several proteins of the same type can be made from one mrna at the same time. 2. Newly synthesised proteins are packaged and sent to Golgi complex for modification/processing. This is called posttranslation processing of the protein Page 6 of 8

7 TYPICAL EXAMINATION QUESTIONS Compare the processes of DNA replication and transcription Similarities Both: (1) involve unwinding the helix (2) involve separating the two strands (3) involve breaking hydrogen bonds between bases (4) involve complementary base pairing (5) involve C pairing with G (6) work in a 5` to 3` direction (7) involve linking/ polymerization of nucleotides (8) DNA or RNA polymerase require a start signal Differences DNA replication Involves DNA nucleotides, where the pentose sugar is deoxyribose, and the base adenine pairs with thymine Both strands are copied Ligase enzyme / no Okazaki fragments are involved Has multiple starting points replication gives two DNA molecules Transcription Involves RNA nucleotides where the pentose sugar is ribose, and the base adenine pairs with uracil Only one strand copied not both No ligase enzyme / no Okazaki fragments Has only one starting point whilst transcription gives mrna Compare DNA transcription with translation Both: (1) Occur in 5 to 3 direction (2) Require ATP Differences (i) DNA is transcribed while mrna is translated (ii) Transcription produces RNA while translation produces polypeptides/ protein (iii) RNA polymerase for transcription while ribosomes for translation/ ribosomes in translation only (iv) Transcription occurs in the nucleus (of eukaryotes) while translation occurs in the cytoplasm/ at ER (v) trna is needed for translation but not transcription Explain briefly the advantages and disadvantages of the universality of the genetic code to humans. (i) Genetic material can be transferred between species/ between humans (ii) One species could use a useful gene from another species (iii) Bacteria/ yeasts can be genetically engineered to make a useful product (1) Viruses can invade cells and take over their genetic apparatus e.g. HIV (2) Viruses cause disease Page 7 of 8

8 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. THE GENETIC CODE CHART / TABLE MOST IMPORTANT PROPERTIES OF GENETIC CODE Property Explanation The nucleotides of mrna are arranged as a linear sequence of codons, each codon 1. The code is a triplet codon consisting of three successive nitrogenous bases, i.e., the code is a triplet codon. In translating mrna molecules the codons do not overlap but are read sequentially. 2. The code is non-overlapping This means that no codon is reserved for punctuations. After one amino acid is coded, 3. The code is commaless the second amino acid will be automatically, coded by the next three letters and that no letters are wasted as the punctuation marks. A particular codon will always code for the same amino acid. The same codon shall 4. The code is non-ambiguous never code for two different amino acids. 5. The code has polarity The code is always read in a fixed direction, i.e., in the 5 3 direction. More than one codon may specify the same amino acid; For example, except for tryptophan and methionine, which have a single codon each, all other 18 amino acids have more than one codon. Biological advantages of degeneracy 6. The code is degenerate (i) It permits essentially the same complement of enzymes and other proteins to be specified by microorganisms varying widely in their DNA base composition. (ii) It provides a mechanism of minimising mutational lethality. E.g. Substitution of the third base-u in GUU (for valine) with C/A/G does not change the amino acid coded for. In most organisms, AUG codon is the start or initiation codon, i.e., the polypeptide 7. Some codes are start codons chain starts either with methionine (eukaryotes) or N-formylmethionine (prokaryotes). Three codons UAG, UAA and UGA are the chain stop or termination codons. They do 8. Some codes are stop codons not code for any of the amino acids. These codons are also called nonsense codons, since they do not specify any amino acid. 9. The code is universal Same genetic code is found valid for all organisms ranging from bacteria to man. Page 8 of 8