Biochemistry I CHM219 Fall 2021 Lecture Notes PDF

BIOCHEMISTRY I CHM219 Assist. Prof. Dr. ESRA AYDEMİR Nucleic Acids  Outline: The Nature of Nucleic Acids Primary Structure of Nucleic Acids Secondary and Tertiary Structure of Nucleic Acids The Biological Functions of Nucleic Acids: A Preview of Molecular Biology Plasticity...

BIOCHEMISTRY I CHM219 Assist. Prof. Dr. ESRA AYDEMİR Nucleic Acids  Outline: The Nature of Nucleic Acids Primary Structure of Nucleic Acids Secondary and Tertiary Structure of Nucleic Acids The Biological Functions of Nucleic Acids: A Preview of Molecular Biology Plasticity of Secondary and Tertiary DNA Structure Stability of Secondary and Tertiary Structure Manipulating DNA The Nature of Nucleic Acids The principal organic constituents of cells and organisms are: Proteins Nucleic acids Carbohydrates Lipids We begin our treatment of biomolecular architecture with nucleic acids due to their roles in storage and transmission of biological information. Chemically, nucleic acid was found to consist of: organic nitrogenous bases a pentose sugar phosphate Later, it was recognized that there are two chemical species of nucleic acid, differing in the nature of the sugar component. DNA (deoxyribonucleic acid) RNA (ribonucleic acid) Both DNA and RNA are polynucleotides. RNA contains the sugar ribose DNA has deoxyribose The two types of heterocyclic bases are derivatives of purine and of pyrimidine. Nucleosides are a nitrogenous base with a ribose. Nucleotides are a nitrogenous base, a ribose and a phosphate. G, T, and U can partially tautomerize to enol forms. A and C tautomerize to imino forms.  Ultraviolet absorption spectra of ribonucleotides. This strong absorbance is often used for quantitative determination of nucleic acids because it allows measurement of nucleic acid concentrations at the microgram/mL level by measurement of light absorption at 260 nm. Nucleic acids are metastable compounds that are thermodynamically favored to break down, but do so only very slowly unless the reaction is catalyzed. The nucleoside monophosphate being added to the growing chain is presented as a nucleoside triphosphate, like ATP or deoxy ATP (dATP), and pyrophosphate is released in the reaction. The reaction is further favored because of the hydrolysis of the pyrophosphate product to orthophosphate, or inorganic phosphate (Pi).  How polynucleotides are actually formed. Each monomer is Formation of a presented as an NTP to polynucleotide by a be added to the chain. hypothetical dehydration reaction. Cleavage of the NTP provides the free energy that makes the reaction thermodynamically favorable. The enzymes catalyzing such reactions are called polymerases. Primary Structure of Nucleic Acids Two important features of all polynucleotides: 1. A polynucleotide chain has a sense or directionality. The phosphodiester linkage between monomer units is between the carbon of one monomer and the carbon of the next. Thus, the two ends of a linear polynucleotide chain are distinguishable. 2. One end normally carries an unreacted phosphate, the other end an unreacted hydroxyl group. A polynucleotide chain has individuality, determined by the sequence of its bases—that is, the nucleotide sequence. This sequence is called the primary structure of that particular nucleic acid.  If we state that we are describing a DNA molecule or an RNA molecule, then most of the structure is understood. We can then abbreviate a small DNA molecule as follows: This notation shows: 1.The sequence of nucleotides, by their letter abbreviations (A, C, G, T). 2.All phosphodiester links are between hydroxyls and phosphates. 3.This particular molecule has a phosphate group at its 5’ end and an unreacted hydroxyl at its 3’ end. 4.It also tells us it is a DNA sequence, not RNA because it has T, not U.  The primary structure of nucleic acids is nearly always written from the 5’ end to the 3’ end. Other shorthand notations are: o pApCpGpTpT o or, more simply o ACGTT The main importance of a DNA sequence is that genetic information is stored in the primary structure of DNA. A gene is nothing more than a particular DNA sequence, encoding information in a four-letter language in which each “letter” is one of the bases. Experiments that (b) Hershey and showed DNA to be Chase showed that the genetic substance. it is the transfer of just the viral DNA (a) Avery et al. showed from a virus to a that nonpathogenic bacterium that pneumococci could be gives rise to new made pathogenic by viruses. transfer of DNA from a pathogenic strain. Secondary and Tertiary Structure of Nucleic Acids A-T and G-C are the base pairs in the Watson–Crick model of DNA. o Note the base pairing occurs between the keto tautomers. o The AT pair has two hydrogen bonds. o The GC pair has three hydrogen bonds. The complementary, two-strand structure of DNA explains how the genetic material can be replicated. This view down the helix axis shows how the base pairs stack on one another, with each pair rotated 36° with respect to the next. The Biological Functions of Nucleic Acids: A Preview of Molecular Biology A side view of the base pairs shows the 0.34-nm distance between the base pairs. This distance is called the rise of the helix. Secondary and Tertiary Structure of Nucleic Acids A space-filling model of DNA. The DNA molecule as modeled by Watson and Crick is shown here with each atom given its van der Waals radius. This model clearly shows how closely the bases are packed within the helix. Note the location of the major and minor grooves.  A model for DNA replication: Each strand acts as a template for a new, complementary strand. When copying is complete, there will be two double-stranded daughter DNA molecules, each identical in sequence to the parent molecule. Three models of DNA replication. Experimental evidence supports the semiconservative model. Brown, parental DNA Blue, new DNA The Meselson–Stahl experiment proves DNA replicates semiconservatively. At pH 7 the DNA is double- stranded. At pH 12 the strands are separated, and DNA is in the random-coil configuration. The two major forms of polynucleotide secondary structure are called A and B. Most DNA is in the B form Double-stranded RNA and DNA-RNA hybrids are in the A form. The 2’ hydroxyl of RNA lies too close to the phosphate and carbon 8 on the adjacent base for RNA to adopt a B form. Comparison of the two major forms of DNA. The structure of B-DNA from studies of molecular crystals. Note the local distortions of the idealized structure previously shown. Many DNA molecules are slightly bent from the vertical axis.  DNA in cells can differ in size and shape. DNA can be from thousands to millions of base pairs in length. DNA can be circular or linear. DNA can be relaxed or supercoiled Viral single-strand DNA (circular) Bacteriophage double-strand DNA (linear)  Relaxed and supercoiled DNA molecules. Electron micrograph showing three human mitochondrial DNA molecules. All three are of identical sequence and contain 16,569 bp each. However, the molecule in the center is relaxed, whereas those at top and bottom are tightly supercoiled. Most DNA molecules found in vivo are left-handed supercoils. Conformations of single-stranded nucleic acids. (a)The random coil structure of denatured single strands. There is flexibility of rotation of residues and no specific structure. (b)Stacked-basostructure adapted by non– self-complementary single strands under “native” conditions. Bases stack to pull the chain into a helix, but there is no H- bonding. (c)Hairpin structures formed by self- complementary sequences; the chain folds back on itself to make a stem–loop structure.  The DNA or RNA sequence is a primary structure, held together by covalent bonds The regular folding patterns observed in the A- and B-DNA double helices are referred to as their secondary structures, held together by non-covalent hydrogen bonds. The high-order folding of DNA’s secondary structure is called its tertiary structure. These structure are also held together by non-covalent interactions.  RNA molecules are usually single-stranded. Most have self-complementary regions that form hairpin structures. Some have well-defined tertiary structures. The Biological Functions of Nucleic Acids: A Preview of Molecular Biology Every organism carries in each of its cells at least one copy of the total genetic information possessed by that organism. This is referred to as the genome. Usually, the genomic information is coded in the sequence of double- stranded DNA, but some viruses use single-stranded DNA or RNA. Genomes vary enormously in size: o The smallest viruses need only a few thousand bases (b) or base pairs (bp). o The human genome consists of about 1 x 109 bp of DNA, distributed in 23 chromosomes.  DNA replication is the copying of both strands of a duplex DNA to produce two identical DNA duplexes. The replication of DNA is accomplished by a complex of enzymes called the replisome. DNA polymerase has multiple functions. As parental DNA strands unwind, forming a replication fork, DNA polymerase guides the pairing of incoming dNTP, each with its complementary partner on the strand being copied. It then catalyzes the formation of the phosphodiester bond to link this residue to the new growing chain.  Each of the parental DNA strands serves as a template, specifying the sequence of a daughter strand. DNA polymerase adds nucleotides, one at a time, to the growing daughter strand, which can be considered a primer to which nucleotides are added as the daughter DNA strand grows from its 5’ end toward its 3’ end. DNA polymerase also “proofreads” the addition before proceeding to add the next residue. This proofreading contributes to the high overall accuracy of replication. Because the two DNA strands run in opposite directions, one daughter strand is elongated in the same direction as that of the replication fork while the other is formed in the reverse direction.  Transcription is the copying of a DNA strand into a complementary RNA molecule. In contrast to DNA replication, in transcription, ribonucleoside triphosphates (ATP, GTP, CTP, and UTP) are needed to make RNA. Note that U in the new RNA pairs with A in the DNA template.) Another distinction from DNA replication is that only one of the two DNA strands, the template strand, is copied.  The linear sequence of bases that constitutes the protein-coding information is “read” by the cell in blocks of three nucleotide residues, or codons, each of which specifies a different amino acid. The set of rules that specifies which nucleic acid codons correspond to which amino acids is known as the genetic code. Complementary copies of the genes to be expressed are transcribed from DNA in the form of messenger RNA (mRNA) molecules.  The basic principle of translation. A mRNA molecule is bound to a ribosome, and tRNA molecules bring amino acids to the ribosome one at a time. Each tRNA identifies the appropriate codon on the mRNA and adds this amino acid to the growing protein chain. The ribosome travels along the mRNA so that the genetic message can be read and translated into a protein. The flow of genetic information in a typical cell. DNA can both replicate and be transcribed into RNA. Messenger RNAs are translated into protein amino acid sequences.  Plasticity of Secondary and Tertiary DNA Structure The forming of a DNA supercoil: Nucleic acid bases can exist in syn or anti conformations.  Z-DNA is a left-handed helix with alternate purine/pyrimidine bases in alternate syn/anti conformation. o Purines are syn. o Pyrimidines are anti. There is now abundant evidence that Z- DNA exists in living cells. The exact role played by Z-DNA in vivo is still an open question.  Self-complementarity in a base sequence allows a chain to fold back on itself and form a base-paired, antiparallel helix or hairpin structure Double hairpins, often called cruciform (cross-like) structures, can be formed in some DNA sequences. To form this structure, the sequence must be palindromic. The word palindrome is of literary origin and usually refers to a statement that reads the same backward and forward, such How self-complementarity as “Able was I ere I saw Elba.” dictates the tertiary structure of tRNA.  Stability of Secondary and Tertiary Structure Denaturation of DNA. a)When native (double-stranded) DNA is heated above its “melting” temperature, it is denatured (separates into single strands). The two randomcoil strands have a higher entropy than the double helix. b)At low T, DG is positive and denaturation of DNA is not favored. As T increases, -TDS overcomes DH, making DG negative and denaturation favorable. The midpoint of the curve marks the “melting” temperature, Tm, of DNA. Denaturation of DNA. c)Absorption spectra of native and denatured DNA show that native DNA absorbs less light than denatured DNA, with the maximum difference occurring at a wavelength of 260 nm. This hypochromicity of doublestranded DNA can be used to distinguish between native and denatured forms. d)The change in absorbance can be used to follow the denaturation of DNA as temperature increases. An abrupt increase in absorbance, corresponding to the sudden “melting” of DNA, is seen at Tm. Baz çifti bileşiminin DNA'nın denatürasyon sıcaklığına etkisi. Grafik, yüzdesi (G + C) arttıkça DNA'nın "erime" sıcaklığındaki artışı göstermektedir. Manipulating DNA Recombinant DNA techniques are used to study nucleic acids and their transcription and translation into proteins Recombinant DNA techniques include: 1. Gene cloning 2. Chemical synthesis of oligonucleotides for use as primers 3. DNA sequence analysis 4. Site-directed mutagenesis 5. Polymerase chain reaction (PCR)  Manipulating DNA – Cloning A clone is a population of organisms that are genetically homogeneous because they were derived from a single ancestor. For example, bacteria on a Petri plate. The first of the developments leading to cloning of single genes was the characterization of plasmids as small circular DNA molecules capable of independent replication within bacterial cells. Clinical resistance to antibiotics is often caused by mutations in genes carried on plasmid DNA molecules. The second development was the discovery of a class of bacterial enzymes called restriction endonucleases and, more important, the fact that many of these enzymes catalyze cleavage of DNA at specific sites. Creation of recombinant DNA molecules in vitro Cloning a fragment of DNA into a plasmid vector and introducing the recombinant molecule into bacteria.  pBR322 is one of the earliest cloning vectors. Some of the restriction sites are shown, as well as the direction of transcription of the ampicillin and tetracycline resistance genes. The bottom diagram shows the effect of cloning a novel sequence into the HindIII site. Manipulating DNA – Chemical Synthesis Many techniques manipulating DNA require the use of primers to initiate replication. The advent of solid-phase DNA synthesis allows for the chemical synthesis of oligonucleotides. One base is added at a time, so any primer that is desired can be synthesized. The synthesis can be automated for the rapid preparation of primers. Solid-phase synthesis of oligonucleotides by the phosphoramidite method.  Manipulating DNA - Sequencing In 1976, Fred Sanger developed a method to determine the sequence of DNA. The technique employed 2’,3’-dideoxynucelotides. Cloning into M13 and sequencing by the Sanger method.