Nucleic Acids and Proteins

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1 DNA Structure 7.1 Nucleic Acids and Proteins Topic 7 DNA is composed of subunits called nucleotides The four types of bases on nucleotides are: Adenine, Guanine, Cytosine, and Thyamine Purines: two ring structure Adenine and Guanine (remember Pure As Gold) Pyrimidines: one ring structure Cytosine and Thyamine pyridimine purine Bases of nucleotides are bonded together by hydrogen bonds A T = 2 H bonds G C = 3 H bonds Sep 4 10:41 PM the phosphates and sugars of the DNA backbone are bonded by a phosphodiester bond Each side has a 3 end and a 5 end The two strands of DNA run anti parallel to each other, > This means that the two backbones run in opposite directions > One has the 5' carbon on top and 3' on the bottom, the other is opposite 1

2 The two antiparallel strands of DNA coil into a double helix structure This double helix further coils up around special proteins called histones DNA wrapped around a set of eight histone molecules is called a nucleosome Nucleosomes help to supercoil chromosomes and help to regulate transcription (because coiled DNA is not accessible by enzymes) Nucleosome consists of 2 molecules each of 4 different histones. DNA wraps twice around these 8 proteins. Histones are positively charged and DNA is negatively charged so they are attracted. Highly repetitive sequences (satellite DNA) constitutes 5 45% of the genome The sequences are typically between 5 and 300 base pairs per repeat and may be duplicated as many as 10 5 times per genome Highly repetitive sequences are sometimes classified as junk DNA as they have no real known purpose (evolutionary leftover; noncoding) Within the genes of eukaryotic DNA there are non coding portions of DNA are called introns and coding portions called exons Introns are cut out by restriction enzymes when RNA is produced The exons are put back together by other enzymes (called ligases) 2

3 DNA Replication 7.2 DNA replication occurs in a 5 to 3 direction 5 end of the free DNA nucleotide is added to the 3 end of the chain of nucleotides which is already synthesized Replication begins at a particular sequence of nucleotides called the origins of replication 7.2 in eukaryotic chromosomes, replication can be initiated at several different points depending on what gene or genes need to expressed, while in prokaryotes the entire genome is replicated at once It is at these points that a special enzyme called helicase splits the DNA into its two antiparallel strands On the strand running in the 5' >3' direction (called the leading strand), another enzyme called DNA polymerase III latches on at one end of the opening, called the replication bubble DNA Polymerase III begins to continuously lay a new DNA strand from free nucleotides in the nucleus At the same time DNA polymerase III is laying new DNA, helicase is continuing to split the strands, thus allowing replication to continue uninterrupted On the opposite strand running 3' >5 (called the lagging strand), replication is not so simple Because new strands have to be laid in the 5' 3' direction, DNA polymerase III cannot lay continuously as it can on the other strand Instead, RNA primase lays short segments of RNA primer nucleotides at many points along the strand When one segment of primer comes in contact with another, DNA polymerase I attaches and replaces the primer with DNA. These segments of DNA are called Okazaki fragments Once the Okazaki fragments have been laid, they are joined by yet another enzyme known as DNA ligase DNA ligase attaches DNA into the gaps between fragments and completes the new strand. a/a/replication/replication_ani.html 3

4 7.3. Transcription Protein Synthesis occurs in two stages: transcription and translation Transcription Occurs in the nucleus Purpose is to create a molecule of messenger RNA carried out in a 5 3' direction (the 5' end of the free RNA nucleotide is added to the 3 end of the RNA molecule which is already synthesized) RNA polymerase binds to parts of the DNA called promoters in order for separation of DNA strands to occur Transcription proceeds as nucleoside triphosphates (type of nucleotide) bind to the DNA template and are joined by RNA polymerase in the 5' to 3' direction. DNA Replication Transcription Translation From DNA DNA mrna To DNA mrna protein Direction 5' 3' 5' 3' 5' 3' Unzipping enzyme helicase RNA polymerase polymerizes nucleotides DNA polymerase RNA polymerase 7.3 Transcription ends when RNA polymerase reaches a termination site on the DNA When it reaches the terminator, the RNA polymerase releases the RNA strand. The two DNA strands are labeled as sense and anti sense Sep 8 3:01 PM The sense strand is the non template strand and has the same base sequence as the mrna with uracil instead of thymine. The antisense strand is the template strand strand being transcribed and has the same base 4

5 7.3.4 In eukaryotes, some editing must occur in order to produce a mature strand of mrna After RNA has been transcribed enzymes will cut out the introns (useless sequences of repetitive nucleotides) and glue the exons (portions that code for protein) back together 7.3 In some viruses (i.e. HIV) RNA is the primary nucleic acid DNA must be transcribed from RNA in order to enter the host cells genome The enzyme that helps to catalyze this process is called reverse transcriptase 7.3 Reverse transcriptase can be used for biotechnology for example, it can make DNA from a section of mature mrna that codes for the gene for human insulin this DNA section can then be spliced into host DNA (eg E. coli) without the introns 7.3 Transcription in Prokaryotes is slightly different than transcription in Eukaryotes due to the fact that they have a circular chromosome The best of example of prokaryotic transcription is the lac operon model: operons are found only in prokaryotes and are sections of the chromosome that code for a particular gene or set of genes as well as a promoter and an operator region (which will be explained momentarily). 7.3 E. Coli bacteria use three enzymes to break down lactose (milk sugar). These three genes are part of one operon the lac operon A regulator gene, located outside of the operon, codes for a repressor protein that binds to the operator region 7.3 Binding of the repressor to the operator prevents RNA polymerase from binding to the promoter region and thus prevents transcription of the three lac enzyme genes However, an isomer of lactose will bind to the repressor protein, and change its shape so it doesn't fit with the operator This allows for the production of the three enzymes Therefore, if there is no lactose (and therefore no isomers of lactose), the repressor protein will prevent the transcription of the enzymes 5

6 Sep 5 1:45 PM Translation 7.4 Translation Takes place on the ribosomes Free ribosomes (in the cytoplasm) synthesize proteins for use primarily within the cell ribosomes on the rough ER synthesize proteins for secretion or for lysosomes Ribosomes consist of two subunits light and heavy (sometimes called the 30s and 50s subunits respectively) the light piece > composed of one rrna and 33 different proteins the heavy piece > composed of three different types of rrna and 45 different protein molecules mrna binding site is between the two ribosomal subunits There are three binding sites for trna on the ribosome: > A site (for trna binding) > P site (for peptide bonding) > E site (exit site) 6

7 Translation occurs in the 5 3 direction during translation, the ribosome moves along the mrna towards the 3 end. The start codon is nearer to the 5 end than the stop codon. Translation consists of three stages: initiation, elongation, and termination Initiation After transcription is complete, mrna goes to the cytoplasm and attaches its 5' end to the small subunit of the ribosome AUG is the start codon because it initiates the translation process > remember: codon is a base triple on the mrna strand The anticodon on one end of a trna molecule is complimentary to a specific codon on the mrna The codon AUG hydrogen bonds to the anticodon of a trna molecule holding the amino acid methionine (called the initiator trna). The initiator trna is now in the A site As the ribosomal subunits slide over mrna the initiator trna moves from the A site to the P site Elongation Another trna (let's call it trna "X") carrying a specific amino acid attaches itself to the next codon at the A site of the larger subunit these two amino acids (methionine and the amino acid on X) now make a peptide bond with each other The initiator trna lets go of methionine, moves to the E site, then leaves the ribosome At the same time, trna X moves from the A site to the P site Another trna molecule (trna "Y") attaches to the codon in the A site The amino acid that is attached to trna Y makes a peptide bond with the amino acid from trna X 7

8 trna X lets go of it s amino acid, moves to the E site and leaves the ribosome trna Y moves to the P site to make room for the next trna to enter the A site The process repeats itself Termination The stop codon is one that does not code for an amino acid and that terminates the translation process the polypeptide is released and the mrna fragments return to the nucleus > These nucleotides are recycled and used for RNA and DNA synthesis trna also is returned to its free state and attaches to its specific amino acid so as to be ready for the translation process when needed. Sep 11 8:18 PM trna specificity Each amino acid has a specific trna activating enzyme that helps trna to combine with its complimentary amino acid. the enzyme has a 3 part active site that recognizes three things: > a specific amino acid > ATP > A specific trna Sep 11 8:10 PM 8

9 The enzyme attaches the amino acid to the 3' end of the trna. The amino acid attachment site is always the base triple CCA. It is important to note that each trna molecule can attach to one specific amino acid, but an amino acid can have a few trna molecules with which is can combine Sep 11 8:13 PM Sep 11 9:10 PM Proteins 7.5 Four levels of protein structure Primary structure the basic order of amino acids in the polypeptide protein chain, before any folding or bonding between amino acid R groups has occurred. Proteins are usually not functional on the primary level, and all proteins have a primary structure. Determines the next 3 levels of protein organization Changing 1 amino acid will alter the structure of the entire protein (sickle cell anemia). Secondary structure the repeated, regular structure protein chains take due to hydrogen bonding between amino acid amine and carboxyl groups. The secondary structure is usually in one of two forms: > alpha helix (similar to a DNA) > beta pleated sheet (similar in form to the corrugations of cardboard). add strength to proteins. 9

10 Sep 11 8:23 PM Tertiary structure complex, threedimensional protein shape resulting from the folding of the polypeptide due to different types of bonds between the amino acids > hydrogen bonds > disulphide linkages (between two sulfur containing amino acids) > ionic bonds (between the negative and positive molecules of amino acid groups) Important for determining the specificity of the proteins known as enzymes. Tertiary Protein Structure Quaternary structure one or more peptide chains bonded together This is the functional form of many proteins, but again, just as not all proteins have secondary or tertiary structure, not all proteins have a quaternary structure. Hemoglobin is an example of a conjugate protein > quaternary structure also includes prosthetic (nonpolypeptide groups) Hemoglobin 10

11 Fibrous vs. Globular Proteins Fibrous proteins are in their secondary structure, which could be in the alpha helix or beta pleated forms made of a repeated sequence of amino acids that can be coiled tightly around in a pattern that makes it a very strong structure Structural proteins Sep 11 8:25 PM Mostly insoluble in water Two examples are keratin (in hair and skin) and collagen (in tendons, cartilage, and bones) Globular Proteins are in their tertiary or quaternary structure, which is folded, creating a globular, threedimensional shape Used mostly for metabolic process Soluble in water Examples include enzymes and hemoglobin Polar vs. Non Polar proteins Non polar amino acids have non polar (neutrally charged) R chains while polar amino acids have R chains with polar groups (charged either positive or negative) 11

12 Proteins with a lot of polar amino acids are hydrophilic and therefore able to dissolve in water Proteins with many non polar amino acids are more hydrophobic and are less soluble in water Some proteins fold so that the hydrophilic amino acids are on the outside and hydrophobic amino acids are on the inside, some fold the other way. Significance: controlling the position of proteins in membranes creating hydrophilic channels through membranes specificity of active sites in enzymes Six functions/types of proteins: #1 Transport An example is hemoglobin, which transports oxygen around the body within a blood cell #2 Support and structure Actin and myosin are examples of muscle proteins but there is protein in skin and bone as well #3 Enzymes (catalyzation of cellular reactions) examples include amylase and trypsin #4 Hormones (cellular communication) examples include insulin and estrogen #5 Antibodies (fight disease) immunoglobulins are produced by b cells (type of white blood cell) #6 Storage Iron is stored in the liver as part of a complex with the protein ferritin Enzymes 7.6 Metabolic pathways consist of chains and cycles of enzyme catalyzed reactions The products of the first reaction, become the reactants of the second reaction, and so on. Different enzymes catalyze each step. Sep 11 9:09 PM 12

13 Induced Fit Model Recall: lock and key model which states that each enzyme has an active site that fits perfectly with one specific enzyme Sep 20 9:03 PM Induced Fit Model: more accurate and modern, states that the active site is slightly different in shape from the substrate the result is that the substrate s chemical bonds are stressed and must therefore break and reform in another way (thus creating the product(s)) this model explains why some enzymes can fit with more than one substrate Enzymes work by lowering the activation energy of a chemical reaction All reactions, either with or without enzymes, need collisions between molecules in order to occur Many molecules have strong bonds holding them together, and as such require powerful collisions at high speed in order to break these bonds However, increasing the rate of collision to a rate at which these reactions would occur would require prohibitive amounts of energy, usually in the form of heat Enzymes, by stressing substrate bonds in such a way that a weaker collision is required to break them, reduce the amount of energy needed to cause these reactions to occur 13

14 Exothermic vs. Endothermic reactions Exothermic reactions are ones that have a net release of energy the energy released as a result of the chemical reaction is greater than the activation energy) Endothermic reactions are ones that have a net intake of energy the activation energy is greater than the amount of energy released as a result of the reaction Competitive Inhibition Competitive & Non competitive Enzyme Inhibition Competitive inhibition occurs when an inhibiting molecule structurally similar to the substrate molecule binds to the active site, preventing substrate binding > the amount of enzyme inhibition depends upon the inhibitor concentration, substrate concentration, and the relative affinities of the inhibitor and substrate for the active site. Example: the inhibition of folic acid synthesis in bacteria by sulfonamide prontosil (an antibiotic) Example: Ethanol is metabolized in the body by oxidation to acetaldehyde, which is in turn further oxidized to acetic acid by aldehyde oxidase enzymes. Normally, the second reaction is rapid so that acetaldehyde does not accumulate in the body. Non competitive Inhibition A drug, disulfiram (Antabuse) inhibits the aldehyde oxidase which causes the accumulation of acetaldehyde with subsequent unpleasant side effects of nausea and vomiting. This drug is sometimes used to help people overcome the drinking habit. Sep 20 9:21 PM 14

15 Non competitive inhibition occurs when an inhibitor molecule binds to a site on the enzyme called its allosteric site (not the active site) and causes a conformational change in its active site, resulting in a decrease in activity Example: nerve gases like Sarin and DFP (diisopropyl fluorophosphate) inhibit the enzyme acetylcholinesterase Sep 20 9:31 PM End product inhibition The product of a series of enzymatic reactions inhibits an enzyme from earlier in the chain For example, in the series of reactions below an accumulation of end product E inhibits the enzyme that helps to convert A to B A > B > C > D > E form of non competitive inhibition because the end product is binding to the allosteric site of enzyme used earlier in the metabolic pathway Example: ATP can inhibit components of glycolysis. Sep 20 10:19 PM 15