7_DNA RNA.pdf

PRINCIPLES OF BIOCHEMISTRY TOPIC 7 DNA The structure shown here is: A. polysaccharide. B. DNA. C. RNA. D. protein. E. lipid. The structure shown here is an example of a monomer unit used to form: A. RNA B. protein C. DNA D. polysaccharide E. lipid NUCLEIC ACID: DNA & RNA ...

PRINCIPLES OF BIOCHEMISTRY TOPIC 7 DNA The structure shown here is: A. polysaccharide. B. DNA. C. RNA. D. protein. E. lipid. The structure shown here is an example of a monomer unit used to form: A. RNA B. protein C. DNA D. polysaccharide E. lipid NUCLEIC ACID: DNA & RNA  DNA DNA is often called the blueprint of life. In simple terms, DNA contains the instructions for making proteins within the cell. Gregor Mendel the "Father of Genetics" performed an experiment in 1857 that led to increased interest in the study of genetics. Gregor Mendel (1822-1884) Frederick Griffith In 1928 a scientist named Frederick Griffith was working on a project that enabled others to point out that DNA was the molecule of inheritance. Griffith's experiment involved mice and two types of pneumonia, a virulent and a non-virulent kind. He injected the virulent pneumonia into a mouse and the mouse died. Next, he injected the non-virulent pneumonia into a mouse and the mouse continued to live. After this, he heated up the virulent disease to kill it and then injected it into a mouse. The mouse lived on. Last he injected non- virulent pneumonia and virulent pneumonia, which had been heated and killed, into a mouse. This mouse died. Why? Griffith thought that the killed virulent bacteria had passed on a characteristic to the non-virulent one to make it virulent. He thought that this characteristic was in the inheritance molecule. This passing on of the inheritance molecule was what he called transformation. Oswald Avery Fourteen years later (1944) a scientist named Oswald Avery continued with Griffith’s experiment to see what the inheritance molecule was. In this experiment he destroyed the lipids, ribonucleic acids, carbohydrates, and proteins of the virulent pneumonia. Transformation still occurred after this. Next, he destroyed the deoxyribonucleic acid. Transformation did not occur. Avery had found the inheritance molecule, DNA! To understand the DNA molecule better scientists were trying to make a model to understand how it works and what it does. In the 1940’s another scientist named Erwin Chargaff noticed a pattern in the amounts of the four bases: adenine, guanine, cytosine, and thymine. He took samples of DNA of different cells and found that the amount of adenine was almost equal to the amount of thymine, and that the amount of guanine was almost equal to the amount of cytosine. Thus, you could say: A=T, and G=C. This discovery later became Chargaff’s Rule. Rosalind Franklin and Maurice Wilkins Two scientists named, Rosalind Franklin and Maurice Wilkins, decided to try to make a crystal of the DNA molecule. If they could get DNA to crystallize, then they could make an x-ray pattern, thus resulting in understanding how DNA works. These two scientists were successful and obtained an x-ray pattern. The pattern appeared to contain rungs, like those on a ladder between strands that are side by side. It also showed an “X” shape indicating that DNA had a helix shape. Crystalline X-ray diffraction pattern from DNA Rosalind Franklin (1920 - 1958) James Watson and Francis Crick In 1953 two scientists, James Watson and Francis Crick, were trying to put together a model of DNA. When they saw Franklin and Wilkin's picture of the X-ray they had enough information to make an accurate model. They created a model that has not changed much since then. Their model showed a double helix with little rungs connecting the two strands. These rungs were the bases of a nucleotide. Problem: How to bond the bases together, and how to solve the problem of the sizes of the bases? Adenine and Guanine were purines having two carbon-nitrogen rings in their structures. Thymine and Cytosine were pyrimidines having one carbon-nitrogen ring in their structure. If DNA were to have its bases pair up so that the purines and the pyrimidines were together, then it would look wobbly and crooked. Watson and Crick then found that if they paired Thymine with Adenine and Guanine with Cytosine, DNA would look uniform = Chargaff's rule! They also found that a hydrogen bond could be formed between the two pairs of bases. Each side is a complete complement of the other. Watson & Crick building the original model of DNA at the Medical Research Council Unit (Cambridge) in 1953 A Person to Praise By using the picture of the crystallized DNA, Watson and Crick were able to put together the model of DNA. Some have speculated that they did not give Rosalind Franklin enough credit for her work; she had certainly made history. Watson and Crick did use the new information very quickly as it is shown by the fact that their paper showing the model of DNA was published in the same issue of Nature as Franklin's picture. Watson and Crick, did, though, use this new information and information from Avery, Chargaff, Griffith, and others. They simply pieced together the puzzle. The Nobel Prize was awarded a few years after the presentation of the model to Watson, Crick, and Maurice Wilkins. Rosalind Franklin did not receive the prize because she had died of cancer by this time. Maurice Wilkins was able to share the prize with Watson and Crick, though, because of his work with Franklin. Her accomplishment should never be forgotten. DNA, or deoxyribonucleic acid, has a crucial role as the chemical carrier of an organism's genes. Each DNA molecule is made up of two very long polymers connected by the bonding of hydrogen atoms and coiled in the shape of a double helix. Each of the two polymers contains many structures called nucleotides, which, in turn, may be further broken down into three parts: - deoxyribose (a five-carbon sugar) - a phosphate group - a nitrogenous base. There are four different nitrogenous bases that might be present: thymine, cytosine, adenine, and guanine. These four bases are the foundation of the genetic code. Sometimes represented as T, C, A, and G, these chemicals act as the cell's memory, instructing it on how to synthesize enzymes and other proteins. These four nucleotides encode everything an organism needs to live and protects this information with incredible accuracy. In a human being, each cell holds 46 separate DNA molecules, each containing, on the average, about 160 million nucleotide pairs, yet this massive amount of information is stored and replicated almost flawlessly. DNA's Backbone ▪ The backbone of the long DNA molecule is quite strong. ▪ It is made up of alternating sugars and phosphates linked through oxygen atoms. ▪ The bonds between these structures are covalent, and therefore difficult to break. ▪ This strong backbone helps guard the genetic information against destruction or mutation Nucleotides consist of 3 components: 1. Nitrogenous base (weakly alkaline) a) Purine b) Pyrimidine 2. Pentose sugar (5 carbon) a) Ribose b) Deoxyribose 3) Phosphate group Nucleotide - building block of polynucleotide DNA gets its name from deoxyribonucleic acid which is a type of nucleic acid. Nucleic acids are made up of polynucleotide chains which are formed by many nucleotides bonded together. Each nucleotide, the basic unit of a polynucleotide chain, is made with three parts: the phosphate, the sugar, and the nitrogenous base. There are two different kinds of sugars in a nucleotide, deoxyribose and ribose. If the polynucleotide chain forms DNA, then the sugars in its nucleotides are deoxyribose while nucleotides containing ribose as its sugar form RNA. There are five different bases in a nucleotide. These bases are adenine, cytosine, guanine, thymine, and uracil. Uracil is only found in RNA, while thymine is only found in DNA. Each base is identified by the first letter in its name. The Nitrogenous Bases The nitrogenous bases connect to the backbone by bonding to the sugars. As they stick out from the long backbone, they attract a complementary base (adenine bonds with thymine, cytosine to guanine), and thus it is through the weak hydrogen bonding of these bases that the two long polymers of the DNA molecule are connected. Because of the nature and shape of these connections, DNA spirals into a double helix, a shape that can be best described as a twisted ladder (the nitrogenous bases would be the rungs).  Only in RNA Only in DNA Thymine and cytosine are known as pyrimidines, while adenine and guanine are purines. Pyrimidine molecules form six cornered rings - purines are a combination of a five cornered ring and a six cornered ring. Purines, then, are the much larger of the two. Because of their sizes, two purines could never bond with each other in a DNA strand because they would be too large, and pyrimidines couldn't because they would be too small to reach each other. This ensures that purines only match up with pyrimidines in the DNA structure and vice- versa. This factor, along with the fact that adenine and thymine must form two hydrogen bonds to be stable while guanine and cytosine must form three, makes the base pairing system an extremely simple and dependable one: the A-T and C-G pairs are the only ones physically possible.  Pentose sugar (in RNA) (in DNA) Each nucleic acid has a 5-carbon (pentose) sugar as a part of its polymer backbone. For DNA this sugar is deoxyribose, while for RNA it's ribose Notice the numbering convention here. For now, since there are no attachments to these sugars, the carbons are numbered 1 through 5. Later, when we attach the base, the sugar carbon numbers will change to 1' through 5'. The difference between ribose and deoxyribose is the presence of the OH on the 2 (2') position. DNA is 2'- deoxy. This is critical for understanding the chemistry of these two polymers.  Nucleic acids (RNA and DNA) are formed by the condensation of nucleotides, catalyzed by polymerases. The bond that is formed is called a phosphodiester bond. Notice that the bonds in this case are formed between the 3' carbon of one sugar and the 5' carbon of the next sugar. ’ Rather than draw out all these structures, shorthand notations are used to designate nucleic acid polymers. Vertical lines represent the sugars and diagonal lines to represent the phosphodiester bonds. More commonly, we need only indicate the order of bases and the direction of the polymer chain. For instance: 5' - AGTCCGATGCAAGCTCG - 3' The two strands are antiparallel. That is one strand goes in the 3' to 5' direction, while its complementary strand goes in the opposite direction. Properties of DNA: The two chains spiral around each other The two chains, composing one double helix, run in opposite directions - antiparallel (5’ - 3’; 3’ - 5’) The sugar - phosphate backbone is located on the outside DNA is composed of two chains of nucleotides The nucleotide bases are projected toward the center of the DNA molecule The phosphate groups give the DNA molecule a negative charge The bases occupy planes that are perpendicular to the main axis of the molecule The bases are stacked one on top of another The two strands are held together by two forces: hydrogen bonding & hydrophobic effect of base stacking The two strands are held together by two forces: a. the energy of hydrogen bonding between the complementary bases (A=T and G=C). b. the hydrophobic effect. While the edges of the base pairs can make hydrogen bond contacts, the planar surfaces are relatively hydrophobic. Therefore, water is less ordered with the bases outside the helix (no base- paired) than when they are inside (base-paired). The hydrophobic effect that produces this arrangement is called base stacking. Properties of DNA helices A Form B Form Z Form RNA-DNA Hybrid Direction of helix rotation right right left right Residues per turn 11 10 12 11 Rotation per residue 33 deg. 36 deg. –30 deg. 33 deg. Rise 0.255 nm 0.34 nm 0.37 nm 0.255 nm Pitch 2.8 nm 3.4 nm 4.5 nm 2.8 nm The helix has three distinct forms: A- form DNA occurs when the amount of water in the surrounding medium is about 75%. The B- form occurs at much higher moisture content, 92%. Z-DNA, or left-handed DNA occurs in regions of high GC content.  The surface features of B-form DNA have distinct major and minor grooves These surface features will become important when we consider how regulatory proteins interact with DNA sequences The relationship between the two chains of the double helix is referred to as complementarity Example: ACGTGGA is complementary to TGCACCT Complementarity is a key factor in nearly all the activities and mechanisms in which nucleic acids are involved The importance of Watson-Crick Proposal Storage of genetic information Self-duplication and inheritance Expression of the genetic message Denaturation Consequences of DNA strands separation Decrease in hydrophobic interactions between DNA bases Changes in electronic nature of DNA bases Increase in UV absorbance Double stranded DNA is a dynamic structure and should never be considered as a static entity. The two strands are held together by non-covalent interactions (hydrogen bonding and base stacking). The energy of these interactions is such that the helix can come apart quite easily at physiological temperatures. If this were not so, gene expression would not be possible at the temperature of living systems. DNA can be heated, and at a certain temperature, the two strands will come apart. We say that the DNA helix has melted or denatured. This transition can be followed by the increase in the absorption of ultraviolet light by the molecule as it goes from helix to random coil (the denatured form). This is called hyperchromicity. DNA Structural Transitions The increase in UV absorbance at 260 nm for the denatured (coil) is used to follow the transition from helix to coil. Here is an example, using Streptococcus pneumoniae DNA: The temperature at the midpoint of the transition is called the melting temperature or Tm. When the GC content increases, Tm also increases WHY? AT base pairs have two hydrogen bonds and GC pairs have three. Therefore, there is more energy in the GC bond. Consequently, the higher the GC content of a molecule, the higher the melting temperature (more energy is necessary to make the helix-coil transition). Which of the following two DNA molecules would have a lower melting temperature (Tm) than the other? DNA Renaturation When denatured DNA is slowly cooled, it regains the properties of the double helix (absorbs less UV light and behaves like normal genetic material) The complementary single-stranded DNA molecules are capable of reassociating, an event termed renaturation, or reannealing Factors of DNA renaturation Ionic strength of the DNA solution Temperature DNA concentration Incubation period Size of the interacting (complementary) DNA molecules Classes of eukaryotic DNA Highly repeated fraction Moderately repeated fraction Non-repeated fraction Once the DNA strands have been melted away from each other, they can be allowed to come back together, to anneal or renature, by lowering the temperature (given the correct ionic strength of the solution). This process is also a kinetic one. The highly repeated sequences (many copies per genome) will renature rapidly, since kinetically any strands can find a match much more quickly. The moderately repeated fraction will have an intermediate time of renaturation. Finally, the unique sequence (e.g., calf non-repetitive fraction) will have the slowest time of renaturation. PRINCIPLES OF BIOCHEMISTRY RNA  RNA DNA contains all the information needed to maintain a cell's processes, but these precious blueprints never leave the protected nucleus. How, then, is all this data transmitted to the body of the cell itself where it may be put to use? The three types of ribonucleic acid (mRNA, tRNA, and rRNA) allow for the communication and translation of genetic information outside of the nucleus.  DIFFERENCES BETWEEN RNA AND DNA 1. The five-carbon sugar in RNA is ribose, while in DNA it is deoxyribose 2. In place of thymine, RNA uses the nitrogenous base uracil (thus the possible pairs are C-G and A-U) 3. While DNA is a double-helix, RNA is almost always a single stranded molecule 4. RNA is significantly shorter than DNA 5. DNA is more stable than RNA  RIBOSOMAL RNA (rRNA) rRNA combines with several proteins to form ribosome - (function as?) Structure of ribosome consists of 2 subunits/components: Eukaryote: 60S and 40S subunits --> 80S ribosome Prokaryote: 50S and 30S subunits --> 70S ribosome Each of this subunit contains one main rRNA and several proteins Eukaryote (mammalian) RNA Large Small subunit subunit  RIBOSOME FUNCTION Ribosome does not carry genetic information (does not code for any proteins!) Main component of ribosome is RNA, i.e., rRNA Involved in the binding of tRNA to mRNA during protein synthesis Ribosome binds to the 5’ terminal end of mRNA and move towards the 3’ terminal end during protein synthesis Cells that are active in protein synthesis (like actively growing cells) contains many ribosomes  MESSENGER RNA (mRNA) Site of protein synthesis (ribosome) is located in the cytoplasm, but genetic information is stored in the nucleus! mRNA carries the genetic information from nucleus to cytoplasm mRNA makes a copy of the information needed to synthesize protein from the DNA Information copied by mRNA will be translated in the cytoplasm by the transfer RNA(tRNA) This process takes place at ribosome in the cytoplasm  SUMMARY OF EXPERIMENTS TO DETERMINE THE GENETIC CODE 1. The genetic code is read in a sequential manner starting near the 5' end of the mRNA. This means that translation proceeds along the mRNA in the 5' ---> 3' direction which corresponds to the N-terminal to C-terminal direction of the amino acid sequences within proteins. 2. The code is composed of a triplet of nucleotides. 3. That all 64 possible combinations of the 4 nucleotides code for amino acids, i.e. the code is degenerate since there are only 20 amino acids. 4. The precise dictionary of the genetic code was determined with the use of in vitro translation systems and polyribonucleotides. The results of these experiments confirmed that some amino acids are encoded by more than one triplet codon, hence the degeneracy of the genetic code. These experiments also established the identity of translational termination codons.  The Genetic Code U C A G U UUU Phe UCU Ser UAU Tyr UGU Cys UUC Phe UCC Ser UAC Tyr UGC Cys UUA Leu UCA Ser UAA End UGA End UUG Leu UCG Ser UAG End UGG Trp C CUU Leu CCU Pro CAU His CGU Arg CUC Leu CCC Pro CAC His CGC Arg CUA Leu CCA Pro CAA Gln CGA Arg CUG Leu CCG Pro CAG Gln CGG Arg A AUU Ile ACU Thr AAU Asn AGU Ser AUC Ile ACC Thr AAC Asn AGC Ser AUA Ile ACA Thr AAA Lys AGA Arg AUG Met ACG Thr AAG Lys AGG Arg G GUU Val GCU Ala GAU Asp GGU Gly GUC Val GCC Ala GAC Asp GGC Gly GUA Val GCA Ala GAA Glu GGA Gly GUG Val GCG Ala GAG Glu GGG Gly  TRANSFER RNA (tRNA) Process of protein synthesis involves translation of genetic information from mRNA at the ribosome Once the type of amino acid is determined, tRNA brings the amino acid to the ribosome to be polymerized Confirms the codon during protein synthesis tRNA is a type of RNA that is the shortest! With a total number of nucleotide ~90 The functions of both tRNA and rRNA are due to their complex secondary and tertiary structures rRNA folding Fold into complex three- dimensional shapes Folding is driven by the formation of regions having complementary base pairs Typical cloverleaf diagram of a tRNA  GENERAL STRUCTURE OF tRNA ‘Clover leaf’ folding: a structure that is stabilized by hydrogen bonds! http://uoitbiology12u2014.weebly.com/protein-synthesis-and-genetic-code.html