Lecture 2: Nucleic Acids PDF

Lecture 2: Nucleic Acids Hunt for the structure of DNA Watson and Crick restricted themselves to what they saw as chemically and biologically reasonable in terms of DNA structure. A breakthrough occurred in 1952, when Erwin Chargaff visited Cambridge and inspired Crick with a description of experime...

Lecture 2: Nucleic Acids Hunt for the structure of DNA Watson and Crick restricted themselves to what they saw as chemically and biologically reasonable in terms of DNA structure. A breakthrough occurred in 1952, when Erwin Chargaff visited Cambridge and inspired Crick with a description of experiments he had published in 1947. Chargaff had observed that the proportions of the four nucleotides vary between one DNA sample and the next, but that for particular pairs of nucleotides adenine and thymine guanine and cytosine the two nucleotides are always present in equal proportions. Discovery of DNA structure 1953: James Watson a Francis Crick derived the structure of DNA based on the following data: Chemical data: Erwin Chargaff principles: the concentration of thymine and adenine is the same the concentration of cytosine and guanine is the same. Physical data: Maurice Wilkins a Rosalind Franklin after exposure of purified DNA molecules to X-rays, there is a characteristic scattering of rays that signal method of arranging DNA components into a helix. Structure of DNA Features of the proposed DNA structure allow: encoding genetic information in the form of ordered bases (denial of Levene´s tetranucleotide theories) replication of DNA molecule is based on complementary pairing of bases. Chemical properties of bases: Spontaneous mutation of DNA bases can also occur by tautomerization: o enol / keto o amino / imino Tautomerization is a net process by which protons are transferred from one site to another by a series of steps in which the solvent is an intermediary. 1 Types of Nucleic acids – DNA DNA forms the genome of prokaryotes, eukaryotes and DNA-viruses gDNA – genomic, mtDNA - mitochondrial, cpDNA –chloroplast, pDNA - plasmid, recDNA - recombinant, rDNA -ribosomal, aDNA – ancient. cDNA (copy DNA, complementary DNA). dsDNA – double-stranded , ssDNA – single-stranded, cccDNA – covalently closed circular, ocDNA - open circle, linDNA – linear. A-DNA, B-DNA, Z-DNA - conformation influenced by sequence and environment. Special forms of DNA – C-DNA, D-DNA and E-DNA. Types of Nucleic acids – RNA RNA - forms the genome of RNA-viruses, in cellular organisms it is a component of ribosomes and perform various functions in the transmission and realization of genetic information. mRNA - mediator, hnRNA - heteronuclear, tRNA - transfer, rRNA -ribosomal, tmRNA – transfer-messenger RNA snRNA – small-nuclear, snoRNA - small nucleolar, scRNA - small cytoplasmic, gRNA - guide, crRNA – CRISPR RNA miRNA, siRNA, shRNA, piRNA ribozyme: an RNA molecule capable of acting as an enzyme. o RNA splicing cleavage (or ligation) of RNA and DNA o Ribosome – peptide bond formation o Viral replication 2 Nucleotide A nucleotide consists of a nitogen-containing base, a five-carbon sugar, and one or more phosphate groups. phosphatic acid (PHOPHATE) pentose (SUGAR) o ribose o deoxyribose organic base (BASE) o purine base adenine guanine o pyrimidine base cytosine thymine uracil Sugar – Pentose ribose in ribonucleic acids (RNA) deoxyribose in deoxyribonucleic acid (DNA) the difference is in the presence or absence of hydroxyl groups on 2 -́ carbon. Bases are attaching to sugar by β-N-glycosidic bond which is a nitrogen- carbon linkage between the 1' nitrogen of pyrimidine bases or 9' nitrogen of purines bases and the 1' carbon of the sugar group. Pyrimidine and Purine bases in Nucleic acids pyrimidine (cytosine, thymine, uracil) purine (adenine, guanine) Phosphates in Nucleic acids 3  attached to 5´ sugar carbon by ester bound, provides the nucleotides with negative charge Nucleosides and Nucleotides in Nucleic acids Nucleotides are phosphorylated Nucleosides. A nucleoside or nucleotide is named according to its nitrogenous base. Deoxyribonucleotides Nucleoside is composed of a base (adenine, guanine, cytosine, thymine) attached to a sugar (deoxyribose). The nucleoside with an attached phosphate group makes it nucleotide. The name of the nucleoside containing the base adenine is deoxyadenosine and if the phosphate group is attached at the carbon numbered 5′ (five prime) then the formal name of the nucleotide is 2′deoxyadenosine 5′-monophosphate (dAMP) 4 Molecule DNA – synthesis Phosphodiester bonds form between the phosphate group attached to the 5′ carbon of one nucleotide and the hydroxyl group of the 3′ carbon in the next nucleotide. Regular alternation of the sugar-phosphatesugar-phosphate motif forms the backbone of the polynucleotide: sugar- phosphate backbone. Chains have chemical polarity: 1 end contains phosphate - 5 -́ end, the other contains hydroxyl group - 3 -́ end. Elongation (synthesis) of polynucleotide chain always runs in the direction 5 ́ – 3.́ Two types of nucleic acid DNA usually double-stranded molecule Strands are bond by hydrogen bonds between base pairs o adenine - thymine o guanine – cytosine RNA usually single-stranded molecule instead of thymine there is uracil. 5 Base pairing Base pairing within strand is due to hydrogen bonds of opposite bases. between two strings = duplex between three strings = triplex between four strings = quadruplex 6 Tautomerization Standard base-pairing arrangements of the canonical nucleotide isomers. Irregular base- pairing arrangements of the tautomers. Tautomerization - process when a nonstationary proton tunnels from a common location to a less-common position within the aromatic ring. When tautomerization occurs during replication, the DNA sequence will be “misread”, and anomalous base-pairing will added. Watson – Crick pairing rules Chargaff´s rules Rule 1: The amount of Adenine ~ equals the amount of Thymine The amount of Guanine ~ equals the amount of Cytosine The amount of purine = the amount of pyrimidine Rule 2: The amount of A+T = amount of G+C This ratio varies among different organisms, but same in different tissues of the same organism. Structure of DNA B-form dsDNA Complementarity of both strands. Distance of the backbone from the axis = 1 nM. Distance of teo bases is 0,34 nM. Antiparallelism = direction of phosphodiester bonds 5'-3' and 3‘-5‘. Planar character of bases. Small and Large groove = places of protein binding to DNA. 7  The bases are oriented inside the double helix to the energetically most advantageous arrangement. One helix turn accounts for 10.5 basepairs. The diameter 2 nm. Winding creates a large and small groove in the helix. Both twin helix strands are antiparalleland fully complementary. Minor and Major groove of DNA The major and minor grooves are opposite each other. Each runs continuously along the entire length of the DNA molecule. They arise from the antiparallel arrangement of the two backbone strands. The grooves are important in the attachment of DNA Binding Proteins involved in replication and transcription. Strands in DNA helix are in antiparallel orientation Two strands of DNA have the same helical geometry however base pairing holds the strands together with opposite polarity. The 5' end of a strand is paired with the base of the end of the other 3‘ end. This anti-parallel orientation, the consequence that adenine and thymine pair with each other and guanine and cytosine pair with each other. Winding of DNA Rosypal (1998): "If we place the thumb of right hand in the direction of the Double helix axis, then other fingers point direction of its ascent/climbing – it is a right-handed double helix. Left-handed double helix corresponds to a similar rule of the left hand. There is an experiment proving the principle underlying the Vester- Ulbricht hypothesis that the primarily left-handed spinning electrons in cosmic rays could have preferentially destroyed lefthanded precursors of DNA, leaving only righthanded DNA. The sculpture illustrates DNA's right-handed double helix. Conformation of dsDNA or dsRNA Conformation = spatial arrangement of the biomacromolecule into a structure, which is the most energy-efficient under the given conditions. 8  DNA conformation depends on: o nucleotide sequence o water content in the environment o ion force of the environment Forms of DNA B-DNA: right-handed, in aqueous solutions and at normal salt concentrations. A-DNA: right-handed, with 11 bp per turn, in dehydrated samples. Z-DNA: left-handed, with 12 pb per turn, occurrence in double helixes GC-rich, function unclear in living systems. A-form Helix is right-handed. Most RNA and RNA-DNA duplex in this form. Shorter, wider helix than B. Deep, narrow major groove not easily accessible to proteins. Wide, shallow minor groove accessible to proteins, but lower information content than major groove. Favored conformation at low water concentrations. Base pairs tilted to helix axis. B-form Helix is right-handed. Most common DNA conformation in vivo. Narrower, more elongated helix than A. Wide major groove easily accessible to proteins. Narrow minor groove. Favored conformation at high water concentrations (hydration of minor groove seems to favor B-form). Base pairs nearly vertical to helix axis. Z-form  Helix has left-handed sense. 9   Can be formed in vivo, given proper sequence and superhelical tension, but function remains obscure.  Narrower, more elongated helix than A or B.  Major "groove" not really groove.  Narrow minor groove.  Conformation favored by high salt concentrations, some base substitutions, but requires alternating purine-pyrimidine sequence.  Base pairs nearly perpendicular to helix axis.  GpC repeat, not single base-pair.  Zigzag backbone due to C sugar conformation compensating for G glycosidic bond conformation. DNA organization Primary structure Primary structure is sequence of bases in the nucleic acid chain and defines the primary structure of DNA or RNA. The sequence of bases is read in a 5′ → 3′ direction, so that you would read the structure in the figure as ACGT Secondary structure The base-pairing of complementary nucleotides gives the secondary structure of a nucleic acid. 10  In a double-stranded DNA or RNA, this refers to the Watson-Crick pairing of complementary strands. In a single-stranded RNA or DNA, the intramolecular base pairs between complementary base pairs determines the secondary structure of the molecule. Tertiary structure  The tertiary structure of a nucleic acid refers to the three-dimensional arrangement of the nucleic acid.  That is, the arrangement of the molecule in space, as in the tertiary structure of tRNA for example. Quaternary structure Quaternary structure refers to the large shapes and structures that can be made by nucleic acids. Organization of dsDNA The DNA double helix may be arranged in space, in a tertiary arrangement of the strands. The two strands of DNA wind around each other. In a covalently closed circular DNA (cccDNA), this means that the two strands can't be separated. In closed circular DNA the strands can't be separated, the total number of turns in a given molecule of cccDNA is a constant, called the Linking Number, or Lk. The linking number of a DNA is a number and has two components,  Twist (Tw), or number of helical turns of the DNA.  Writhe (Wr), number of times the double helix crosses over on itself - these are the supercoils. Because Lk is a constant, the relationship can be shown by the equation: Lk = Tw + Wr The simplest supercoil - writhe, is the shape a circular DNA assumes to accommodate one too many or one too few helical twists. The two lobes of the figure eight will appear rotated either clockwise or counterclockwise with respect to one another, depending on whether the helix is over or underwound. Twist and writhe are interconvertible. Extra helical twists are positive and lead to positive supercoiling, while subtractive twisting causes negative supercoiling. Normally, this DNA would have a linking number equal to 25, so it is underwound. The DNA double helical structures have the same value of Lk; however, the DNA can be supercoiled, with the two “underwindings” taken up by the negative supercoils. This is equivalent to two “turns‘-writhes” of single-stranded DNA and no supercoils. 11 As a general rule, the DNA of most organisms is negatively supercoiled. DNA – topoisomerases DNA – topoisomerases - alter Lk, the linking number of a DNA, by a bond breaking and rejoining process. Catalyze the formation of transitional breaks in DNA Break - "nick" = breaking of the phosphodiester bond between neighboring bases. DNA – topoisomerase I Type I topoisomerases - sometimes called “nicking-closing enzymes” carry out the conversion of negatively supercoiled DNA to relaxed DNA in increments of one turn. Type I , increases Lk by increments of one to a final value of zero. Type I topoisomerases are energy independent, because they don't require ATP for their reactions. Anti-tumor drugs, including Camptothecin, target the eukaryotic topoisomerase I. Releases superhelix tension from the superhelix DNA. Topoisomerase covalently attaches to one of the phosphates in DNA, o cuts DNA strand can rotate around its longitudinal axis, o the strand tension/pressure is relieved o double helix restoration and enzyme is released. DNA – topoisomerase II - DNA Gyrase Type II topoisomerases (sometimes called DNA gyrases) reduce Lk by increments of two. These enzymes are ATP-dependent and will alter the linking number of closed circular DNA. 12  TOP2 functions by relieving supercoils generated as a function of the double stranded nature of DNA. Type II topoisomerases act on naturally occurring DNAs to make them supercoiled. The enzyme unknots and untangles DNA by passing an intact helix through a transient double-stranded break that it generates in a separate helix. Differences between topoisomerases I and II the one strand moves above the other one in the cut and merges: o L is reduced by 1 both strands move above the break and merges: o + ATP for function o L is reduced by 2 DNA organization in regulation of transcription Eukaryotic DNA is linear and double stranded. It binds to protein scaffolding. Generates superhelixes, solenoid loops and relaxed areas. DNA sequences adopting alternative structures  Unique DNA sequence:....AATGCTGATGTCTGACTCGGA...  Repetitive sequences or repeat.  Terms: unit of repetition, length of unit of repetition, frequency of repetition.  Example: ATG… ATG….ATG….ATG…unit = ATG, length = 3 nucleotides, frequency = 4x. o Tandem repeats – tied tightly to each other "head to toe".. ATGCATGCATGC.. 13  o Direct repetition (5.́...ATGC..... ATGC.....3 )́ repeats on the same strings in the same direction (5 ́ 3 )́.  Inverted repetition: repeated on the second string in the reverse direction - potential for creating a hairpin or hairpin with a loop. Denaturation and Renaturation of DNA and RNA DNA is a highly stable molecule, the non-covalent hydrogen bonds between 2 complementary strands can be broken and the DNA strands can be separated. This is called denaturation.  Denaturation of dsDNA = transformation of dsDNA into ssDNA.  Renaturation of ssDNA = transformation of ssDNA into dsDNA. Induction of denaturation:  by increasing the temperature of the solution  by changing the pH from neutral to alkaline or acidic. Occurs in vitro and naturally also in vivo. dsDNA denaturation is manifested by hyperchromic effect, which means increased absorbance of UV-light with a wavelength 260 nm. Value Tm or melting point = temperature, in which 50% of dsDNA molecules are denatured.  Tm depends on the content of the bases. Other options for determining %GC:  Ultracentrifugation in CsCl  HPLC.  GC = molar fraction of guanine and cytosine in DNA  69.3 and 0.41 are empirically the coefficients laid down  pro poly(AT) Tm = 69,3. 14 Hybridization of DNA and RNA Hybridization is the process of combining two complementary single-stranded DNA or RNA molecules and allowing them to form a single double- stranded molecule through base pairing. The more hybridizing molecules coincide in sequences, or the higher their sequential homology, the greater it is the probability of their hybridization. Use for evaluation of the degree of sequential/structural similarity of DNA without sequencing. 1. Double-helical DNA from two species is unwound (denatured), cut, and mixed. 2. Complementary pieces bind (called reannealing), forming some hybrid DNAs—one strand from each species. 3. Then the temperature is raised and the rate of interspecies double-helix separation determined. The higher the temperature needed to separate the hybrid DNA, the more similar the DNA sequences must be. Usage of Hybridization in the reasearch Identification of specific DNA a RNA sequences. Estimation of their structural similarity. PCR. Transcription in vitro. FISH. 15