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NUCL EIC A CIDS Nucleic acids are involved in the storage and transfer of genetic information in all living organisms, including the simplest viruses. There are 2 types of nucleic acid in cells, Deoxyribonucleic acid (DNA) and Ribonucleic acid (RNA). Nucleic acids are so named because DNA was 1st isolated from nuclei, but both DNA and RNA also occur in other parts of the cell, e.g. DNA is also found in mitochondria and chloroplasts, whilst RNA is also found in the cytoplasm, particularly at the ribosomes. Both DNA and RNA are polymers, the monomeric units being called nucleotides. DNA and RNA are therefore polynucleotides. THERE ARE FIVE DIFFERENT NITROGEN-CONTAINING ORGANIC BASES PRESENT IN NUCLEIC ACIDS: adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U). A, T, C and G are found in DNA. A, U, C and G are found in RNA (uracil replaces thymine here). THE NITROGENOUS BASES BELONG TO TWO DIFFERENT CHEMICAL FAMILIES. Adenine and guanine are purine bases and have a double-ringed structure. Cytosine, thymine and uracil are pyrimidine bases and have a single-ringed structure The combination of a sugar and a base forms a compound called a nucleoside. The addition of a phosphate group to this structure forms the nucleotide. Different nucleotides are formed according to the sugar and bases used. Cells continuously produce nucleotides and these form a "pool" from which nucleotides can be used up as required for manufacturing DNA, RNA or a variety of other substances. DISTINGUISH BETWEEN THE FOLLOWING TERMS: Nucleotide and Polynucleotide Purine and Pyrimidine bases Structure of DNA 1 DNA is a double stranded molecule. It consists of two polynucleotide chains held together by interactions between pairs of bases projecting towards each other from each strand. The interactions involve H-bonding between base pairs. 2. The two polynucleotide chains in a molecule of DNA are not identical but are complementary. The bases always pair up in a specific fashion; adenine always pairs with thymine, cytosine always pairs with guanine. i.e. a purine base pairs with a pyrimidine base. There are several pieces of evidence that support this: a) In any molecule of DNA, the ratio of A:T is always 1:1, and the ratio of C:G is similarly always 1:1. However, the ratio of A or T to C or G is variable between different molecules of DNA. b) Hydrogen bonding can only occur between A and T and between C and G. c) Base pairing between a purine (double-ringed) and a pyrimidine (single-ringed) allows a constant base pair length. It therefore follows that the distance between the two polynucleotide chains will also be constant throughout the length of the molecule. The double-stranded molecule is coiled up into a helical structure with ten nucleotides per turn of the helix. Structure of RNA RNA is a single-stranded polynucleotide. There are three different types of RNA in the cells, each with a particular structure and function. 1.Messenger RNA (mRNA) They are single-stranded and are made up of hundreds to several thousand nucleotides. mRNA is made in the nucleus from coded instructions in the DNA and then passes into the cytoplasm where it is involved in the process of protein synthesis on the ribosomes. 2.Transfer RNA (tRNA) They are single strands of 75-90 nucleotides wound up to form an overall "clover-leaf" shape. All cells have at least twenty different kinds of tRNA. They interact with mRNA during protein synthesis on the ribosomes. Two important features of the tRNA molecules are: a) they possess an `anticodon' loop through which they can interact with molecules of mRNA. b) at the opposite end of the molecule they possess an amino acid binding site. The amino acids carried by tRNA molecules eventually form polypeptide chains during protein synthesis. 3.Ribosomal RNA (rRNA) This is made inside the nucleus within the nucleoli and is a major component of ribosomes. The precise configuration of rRNA is unknown but they are very large molecules containing thousands of nucleotides. DNA REPLICATION A major requirement of genetic material is that it should be able to replicate so that its messages can be passed on from cell to cell as an organism develops and also from one generation to the next. It is also vital that during replication, identical copies of DNA are made so that the correct genetic messages are passed on. DNA replication takes place during interphase of the cell cycle, so that by the time nuclear division starts, two identical copies of each DNA molecule are already present for distribution into daughter cells. Two models of DNA replication have been proposed. Conservative mechanism Generation 1 Generation 2 Make drawings to predict the molecules formed in Generation 3 This model suggests that an entire new helix is synthesised along the original molecule. Therefore the parental molecule is "conserved". Semi-conservative mechanism This model suggests that the DNA double helix gradually "unzips" to expose the bases of each strand. New nucleotides align themselves in a complementary fashion against the bases of each parental strand. These nucleotides are joined by an enzyme (DNA POLYMERASE) to make new polynucleotides. Therefore in the two new double helices, one strand is the original parental strand, whilst the other is newly made; i.e.. only half the parental molecule is conserved in each daughter molecule. Semi-conservative mechanism Generation 1 Make drawings to predict the molecules formed in Generation 3 Generation 2 The following experiment carried out by Meselsohn and Stahl (1957) identified the correct model. The bacterium E.coli was transferred from a growth medium containing normal nitrogen 14N, to a medium containing the heavy isotope of nitrogen 15N. They were grown on this medium for a sufficient number of generations so that all the nitrogenous bases in their DNA contained this heavy isotope. Light and heavy DNA can easily be distinguished by special centrifugation techniques where heavy DNA sediments to form a band lower down the centrifuge tube than light DNA. The bacteria that had been grown in 15N medium were then transferred back to 14N medium and grown for one generation. A sample of cells was taken, the DNA extracted and centrifuged to see if it was light or heavy. It was found that neither type was present. Instead an intermediate band was formed. The cells were left to grow on 14N medium for a second generation and the DNA samples as before. This time, two bands were seen, light and intermediate. Results of the Meselsohn and Stahl experiment Cells grown in 14N Cells transferred from `B' and grown in 14N for Cells grown in 15N 1 2 generations Light DNA Intermediate DNA Heavy DNA A B C D INTERPRET THESE RESULTS AND STATE WHICH MODEL OF DNA REPLICATION IS CORRECT. THE GENETIC CODE DNA is the hereditary material responsible for all the characteristics of an organism and it controls all the activities of a cell. It is able to do this as it carries messages which control the synthesis of proteins. An important class of proteins is the enzymes which control chemical reactions within the cell, including the synthesis and breakdown of other classes of molecule. Therefore, by controlling which proteins are made at a particular time in a particular type of cell, DNA is able to control all the characteristics of a cell. Proteins are made up of amino acids. There are about 20 different types of amino acids commonly found in proteins. The precise number and sequence of amino acids makes up the primary structure of a polypeptide chain. A functional protein may consist of a single, or several polypeptide chains. DNA must therefore carry a coded message that determines not only the number and types of amino acids that appear in a polypeptide, but also their precise sequence in the chain. The code for primary structure cannot be carried in the sugar-phosphate backbone of DNA since this structure is identical in all DNA molecules. The only part of DNA that varies between different molecules is the base sequence. Therefore, the sequence of bases in DNA must determine the sequence of amino acids in a polypeptide chain. The length of DNA that codes for a polypeptide chain is called a gene and it can be thousands of nucleotides long. The code cannot be as simple as 1 base coding for 1 amino acid as this would allow for the coding of only 4 amino acids. If the bases are read together in pairs, this would allow for only 16 different combinations i.e.. 16 different amino acids. Each amino acid is in fact coded for by a sequence of 3 consecutive nucleotide bases in the DNA chain. This is called the triplet base hypothesis and each triplet of bases is called a codon. The maximum number of combinations this allows is 64, but since there are only 20 common amino acids, some are coded for by more than one triplet codon. The genetic codon is thus described as degenerate. The genetic code is usually represented in the form of RNA that would be complementary to the DNA in the gene. This is because it is messenger RNA that is directly involved in protein synthesis and not the genes themselves. Some of the triplet codons do not code for any amino acid. These are called non-sense codons and they act as signals to terminate the synthesis of the polypeptide chain i.e.. they act as "full stops" and are called termination codons. Some other triplets code for modified amino acids that act as signals to start the synthesis of the protein chain and these are called initiation codons. The Genetic Code Each triplet of bases represents a sequence in mRNA. Each sequence codes for the amino acid shown. 2nd base of codon U U C A G C A G UUU UUC UUA UUG Phe Phe Leu Leu UCU UCC UCA UCG Ser Ser Ser Ser UAU UAC UAA UAG Tyr Tyr *Term *Term UGU Cys UGC Cys UGA *Term UGG Try U C A G CUU CUC CUA CUG Leu Leu Leu Leu CCU CCC CCA CCG Pro Pro Pro Pro CAU CAC CAA CAG His His Gln Gln CGU CGC CGA CGG Arg Arg Arg Arg U C A G AUU AUC AUA AUG Ile Ile Ile Met *Init ACU ACC ACA ACG Thr Thr Thr Thr AAU AAC AAA AAG Asn Asn Lys Lys AGU AGC AGA AGG Ser Ser Arg Arg U C A G GUU GUC GUA GUG Val Val Val Val *Init GCU GCC GCA GCG Ala Ala Ala Ala GAU GAC GAA GAG Asp Asp Glu Glu GGU GGC GGA GGG Gly Gly Gly Gly U C A G AMINO ACIDS Phe = phenylalanine Ile = isoleucine Ser = serine Thr = threonine His = histidine Cys = cysteine Try = tryptophan Arg = arginine Gly = glycine Val = valine Leu = leucine Met = methionine Pro = proline Ala = alanine Tyr = tyrosine Gln = glutamine Asn = asparagine Lys = lysine Asp = aspartic acid Glu = glutamic acid TRIPLETS MARKED: * Init = initiation of polypeptide *Term = termination of polypeptide PROTEIN SYNTHESIS Transcription This is the first stage in protein synthesis. The genetic information required for protein synthesis is contained in DNA which remains in the nucleus. Protein synthesis however, occurs at the ribosomes in the cytoplasm (rough endoplasmic reticulum). Therefore, a messenger molecule (mRNA) is made, which carries the required genetic information from the nucleus to the ribosomes. The process by which mRNA is made is called transcription (or DNA-dependent RNA synthesis.) The DNA double helix unwinds in that part which carries the code for the required protein (the gene). One of the strands then acts as a template (pattern) for the production of mRNA. mRNA synthesis occurs in 3 stages: mRNA synthesis involves the association of the enzyme RNA polymerase with the DNA template. Therefore, mRNA carries an identical genetic message to the gene, but in a complementary nucleotide sequence. The mRNA then passes through pores in the nuclear membrane to the ribosomes where one end of the molecule becomes attached to a ribosome. The information carried by mRNA must now be decoded to form a protein. Translation This is the process by which the genetic information in mRNA directs the synthesis of a polypeptide by controlling the order of insertion of amino acids into the growing polypeptide (i.e.. it determines the protein's primary structure). It occurs in four stages: This involves a species of RNA called transfer (t) RNA. Its function is to decode the message carried by mRNA by correctly positioning amino acids into the growing polypeptide chain according to the sequence of nucleotides in mRNA. Each cell contains about 60 different types of tRNA it is single stranded, 70-90 nucleotides long, and portions of the molecule are wound up into a double helix to give a clover-leaf shape. Each tRNA molecule possesses three important features: 1 An anticodon site, which consists of a triplet of unpaired bases. The sequence of bases in this site varies from molecule to molecule and there is an anticodon sequence that is complementary to each codon sequence found on mRNA. 2 An attachment site at the free end of the molecule that can bind a specific amino acid. 3 A recognition site that enables the correct amino acid to bind to each tRNA molecule. The particular amino acid that binds to each tRNA molecule is somehow determined by the anticodon sequence. The actual amino acid is that which would be specified by the nucleotide sequence complementary to the anticodon, i.e. the codon on mRNA. E.g.. the codon UCU specifies the amino acid serine. Thus the tRNA molecule that could recognise and bind serine would carry the anticodon AGA. However, before the correct amino acid can be bound to tRNA, it must first undergo an initial activation step. Twenty amino acids are commonly involved in protein synthesis. Each amino acid of the common 20, has its own specific activation enzyme which forms a complex with its specific amino acid. Energy is required for this and is provided by ATP. The activated amino acid is then accepted by a specific tRNA molecule to form a complex called an amino acyl tRNA. Consider a sequence of mRNA to be translated: AUGAAACGGUUA mRNA codons for: *met lys arg leu amino acids *met = methionine (initiation) arg = arginine lys = lysine leu = leucine 1 An mRNA molecule attaches to a ribosome. A tRNA molecule with an attached a.a. bearing the anticodon to the codon AUG and a modified amino acid (a modified molecule of the amino acid methionine) is positioned on the ribosome. This marks the initiation of the polypeptide chain. (also energy is required.) The ribosome is now ready to receive the tRNA with attached a.a. specified by the next codon, AAA, i.e.. the tRNA with attached a.a bearing the anticodon UUU. 2 A peptide linkage is now formed between the two adjacent amino acids. The amino acid becomes detached from its tRNA, and a dipeptide is now attached to the tRNA which is still at the ribosome. 3 The next tRNA molecule (bearing the anticodon GCC and the amino acid arginine) is positioned. Energy is required for the selection and binding of tRNA molecules. Once again, a peptide linkage is formed between the amino acids and in this way, the peptide increases in length. 4 These steps are repeated until the entire sequence of codons on mRNA have been "read". Termination of protein synthesis occurs when a termination codon (UAA,UAG or UGA) is reached and the newly synthesised polypeptide is released from the final tRNA molecule. Following completion, the polypeptide may undergo modification before it becomes a functional protein In eukaryotic cells, the polypeptide is released into cavity of the RER for transport through the cell. Often several ribosomes "read" simultaneously along the same messenger so that several polypeptides can be made from the same mRNA. A chain of ribosomes attached to the same mRNA is called a polysome. Protein synthesis is an energy consuming process. Energy, in the form of ATP is required for: chain initiation amino acid activation translocation selection and binding of amino acyl tRNAs to the A site The role of nucleic acids in the storage and transfer of genetic information can be summarised as follows: transcription translation DNA RNA Proteins Metabolic reactions and their regulation SOME SAMPLE QUESTIONS: Nucleic acids 1 Distinguish between the following: nucleotide and polynucleotide (2) 2 List FIVE differences between DNA and RNA. (5) 3 The following is the sequence of bases in one of the two strands of part of a DNA molecule. CAGGTACTG (a) 4 What will be sequence of bases in the complementary strand? (1) The following sequence of bases in DNA codes for the formation of a short peptide chain: TACTTTAGAGGACCAGTAATT (a) (b) 6 Show the sequence of bases you would expect to find in the corresponding messenger RNA molecule. (1) What will be the resulting sequence of amino acids in the finished peptide chain? (use code table in h/o) (1) Lysozyme is a protein made up of 129 amino acids. (a) (b) (c) How many DNA nucleotides are needed to encode for this chain of amino acids? (1) A complete turn of the DNA double helix contains 10 pairs of bases and is 3.4 nm long. What length of DNA molecule is occupied by the gene for lysozyme? (1) How many turns of the DNA double helix does this represent? (1)