Nucleotides and Nucleic Acids Chapter 19 Nucleotides and Nucleic Acids 1

Information Transfer in Cells Information encoded in a DNA molecule is transcribed via synthesis of an RNA molecule The sequence of the RNA molecule is "read" and is translated into the sequence of amino acids in a protein. 3

Know the basic structures Nitrogenous Bases Know the basic structures Pyrimidines Cytosine (DNA, RNA) Uracil (RNA) Thymine (DNA) Purines Adenine (DNA, RNA) Guanine (DNA, RNA) Cytosine (C) Thymine (T) Uracil (U) DNA & RNA DNA RNA Adenine (A) Guanine (G) DNA & RNA 4

Properties of Pyrimidines and Purines Keto-enol tautomerism 5

Properties of Pyrimidines and Purines Acid/base dissociations Strong absorbance of UV light 5

Pentoses of Nucleotides Know these structures too D-ribose (in RNA) 2-deoxy-D-ribose (in DNA) The difference - 2'-OH vs 2'-H This difference affects secondary structure and stability 6

Linkage of a base to a sugar 11.3 Nucleosides Linkage of a base to a sugar Base is linked via a glycosidic bond The carbon of the glycosidic bond is anomeric 7

Linkage of a base to a sugar 11.3 Nucleosides Linkage of a base to a sugar Named by adding -idine to the root name of a pyrimidine or -osine to the root name of a purine 7

Linkage of a base to a sugar 11.3 Nucleosides Linkage of a base to a sugar Conformation can be syn or anti Sugars make nucleosides more water-soluble than free bases 7

Nucleoside phosphates 11.4 Nucleotides Nucleoside phosphates Nucleotides are nucleosides esterified with phosphoric acid Most are esterified at the 3’ or 5’ position "Nucleotide phosphate" is redundant! Nucleotides are poly-protic acids 8

Nucleoside phosphates 11.4 Nucleotides Nucleoside phosphates 8

Functions of Nucleotides Nucleoside 5'-triphosphates are carriers of energy Bases serve as recognition units 9

Functions of Nucleotides Cyclic nucleotides are signal molecules and regulators of cellular metabolism and reproduction ATP is central to energy metabolism GTP drives protein synthesis CTP drives lipid synthesis UTP drives carbohydrate metabolism 9

11.5 Nucleic Acids - Polynucleotides Following are names and one-letter abbreviations for the heterocyclic aromatic amine bases most common to nucleic acids 10

11.5 Nucleic Acids - Polynucleotides Polymers linked 3' to 5' by phosphodiester bridges Ribonucleic acid (RNA) and deoxyribonucleic acid(DNA) Know the shorthand notations Sequence is always read 5' to 3' In terms of genetic information, this corresponds to "N to C" in proteins T-G is a dinucleotide 10

11.5 Nucleic Acids - Polynucleotides 10

11.5 Nucleic Acids - Polynucleotides 10

11.6 Classes of Nucleic Acids DNA - one type, one purpose RNA - 3 (or 4) types, 3 (or 4) purposes ribosomal RNA - the basis of structure and function of ribosomes messenger RNA - carries the message transfer RNA - carries the amino acids 11

Structure of DNA Primary Structure: the sequence of bases along the pentose-phosphodiester backbone of a DNA molecule (or an RNA molecule) read from the 5’ end to the 3’ end Secondary structure: the ordered arrangement of nucleic acid strands Double helix: a type of 2° structure of DNA molecules in which two antiparallel polynucleotide strands are coiled in a right-handed manner about the same axis

Stabilized by hydrogen bonds! The DNA Double Helix Stabilized by hydrogen bonds! "Base pairs" arise from hydrogen bonds Erwin Chargaff had the pairing data, but didn't understand its implications Rosalind Franklin's X-ray fiber diffraction data was crucial Francis Crick knew it was a helix James Watson figured out the H-bonds 12

DNA - 2° Structure T-A base pairing

DNA - 2° Structure C-G base pairing

DNA - 2° Structure Ribbon model of B-DNA

DNA - 2° Structure B-DNA the predominant form in dilute aqueous solution a right-handed helix 20 Å thick with 34 Å per ten base pairs minor groove of 12 Å and major groove of 22 Å A-DNA a right-handed helix, but thicker than B-DNA 29 Å per 10 base pairs Z-DNA a left-handed double helix

The Structure of DNA Length of 1.6 million nm (E. coli) Compact and folded (E. coli cell is only 2000 nm long) Eukaryotic DNA wrapped around histone proteins to form nucleosomes 13

DNA - 3° Structure Tertiary structure: the three-dimensional arrangement of all atoms of a nucleic acid, commonly referred as to supercoiling Circular DNA: a type of double-stranded DNA in which the 5’ and 3’ ends of each stand are joined by a phosphodiester bond (Fig 20.10) Chromatin: consists of DNA molecules wound around particles of histones in a beadlike structure

DNA - 3° Structure Relaxed, strained, and supercoiled DNA

Ribonucleic Acids (RNA) RNA are similar to DNA in that they, too, consist of long, unbranched chains of nucleotides joined by phosphodiester bonds between the 3’-OH of one pentose and the 5’-OH of the next; however: the pentose unit in RNA is -D-ribose rather than -2-deoxy-D-ribose the pyrimidine bases in RNA are uracil and cytosine rather than thymine and cytosine RNA is single stranded rather than double stranded

RNA RNA molecules are classified according to their structure and function Ribosomal RNA (rRNA): a ribonucleic acid found in ribosomes, the site of protein synthesis

RNA Transfer RNA (tRNA): a ribonucleic acid that carries a specific amino acid to the site of protein synthesis on ribosomes

RNA Messenger RNA (mRNA): a ribonucleic acid that carries coded genetic information from DNA to the ribosomes for the synthesis of proteins present in cells in relatively small amounts and very short-lived single stranded their synthesis is directed by information encoded on DNA a complementary strand of mRNA is synthesized along one strand of an unwound DNA, starting from the 3’ end

RNA the synthesis of mRNA from DNA is called transcription

Genetic Code

Genetic Code Properties of the Code only 61 triplets code for amino acids; the remaining 3 (UAA, UAG, and UGA) signal chain termination the code is degenerate, which means that several amino acids are coded for by more than one triplet; Leu, Ser, and Arg, for example, are each coded for by six triplets for the 15 amino acids coded for by 2, 3, or 4 triplets, it is only the third letter of the codon that varies; Gly, for example, is coded for by GGA, GGG, GGC, and GGU there is no ambiguity in the code; each triplet codes for one and only one amino acid

Sequencing DNA Restriction endonuclease: an enzyme that catalyzes hydrolysis of a particular phosphodiester bond within a DNA strand over 1000 endonucleases have been isolated and their specificities determined typically they recognize a set sequence of nucleotides and cleave the DNA at or near that particular sequence EcoRI from E. coli, for example, cleaves as shown

Sequencing DNA examples of restriction endonucleases

Sequencing DNA Polyacrylamide gel electrophoresis: a technique so sensitive that it is possible to separate nucleic acid fragments differing from one another in only a single nucleotide Chain termination or dideoxy method: a method developed by Frederick Sanger for sequencing DNA molecules

DNA Replication the sequence of nucleotides on one strand is copied as a complementary strand to form the second strand of double-stranded DNA this synthesis is catalyzed by the enzyme DNA polymerase DNA polymerase will carry out this synthesis in vitro using single-stranded DNA as a template, provided the four dNTPs and a primer are present because the new DNA strand grows from the 5’ to 3’ end, the primer must have a free 3’-OH group to which the first nucleotide of the growing chain is added

Chain-Termination Sequencing the key is addition of a 2’,3’-dideoxynucleoside triphosphate (ddNTP) to the synthesizing medium synthesis terminates at any point where a ddNTP becomes incorporated

Chain-Termination Sequencing a single-stranded DNA of unknown sequence is mixed with primer and divided into four separate reaction mixtures to each mixture is added all four dNTPs, one of which is labeled in its 5’- phosphoryl group with P-32 also added are DNA polymerase and one of the four ddNTPs when polyacrylamide gel electrophoresis of each reaction mixture is completed, a piece of x-ray film is placed over the gel to detect gamma radiation from the decay of P-32 the base sequence of the complement to the original single-stranded template is read directly from the bottom to top of the developed film

Transcription product of DNA Messenger RNA Transcription product of DNA In prokaryotes, a single mRNA contains the information for synthesis of many proteins In eukaryotes, a single mRNA codes for just one protein, but structure is composed of introns and exons 14

Eukaryotic mRNA DNA is transcribed to produce heterogeneous nuclear RNA mixed introns and exons with poly A intron - intervening sequence exon - coding sequence poly A tail - stability? Splicing produces final mRNA without introns 15

Ribosomal RNA Ribosomes are about 2/3 RNA, 1/3 protein rRNA serves as a scaffold for ribosomal proteins 23S rRNA in E. coli is the peptidyl transferase! 16

Transfer RNA Small polynucleotide chains - 73 to 94 residues each Several bases usually methylated Each a.a. has at least one unique tRNA which carries the a.a. to the ribosome 3'-terminal sequence is always CCA-a.a. Aminoacyl tRNA molecules are the substrates of protein synthesis 17

Why does DNA contain thymine? DNA & RNA Differences? Why does DNA contain thymine? Cytosine spontaneously deaminates to form uracil Repair enzymes recognize these "mutations" and replace these Us with Cs But how would the repair enzymes distinguish natural U from mutant U? Nature solves this dilemma by using thymine (5-methyl-U) in place of uracil 18

Why is DNA 2'-deoxy and RNA is not? DNA & RNA Differences? Why is DNA 2'-deoxy and RNA is not? Vicinal -OH groups (2' and 3') in RNA make it more susceptible to hydrolysis DNA, lacking 2'-OH is more stable This makes sense - the genetic material must be more stable RNA is designed to be used and then broken down 19

Hydrolysis of Nucleic Acids RNA is resistant to dilute acid DNA is depurinated by dilute acid DNA is not susceptible to base RNA is hydrolyzed by dilute base See Figure 11.29 for mechanism 20

Restriction Enzymes Bacteria have learned to "restrict" the possibility of attack from foreign DNA by means of "restriction enzymes" Type II and III restriction enzymes cleave DNA chains at selected sites Enzymes may recognize 4, 6 or more bases in selecting sites for cleavage An enzyme that recognizes a 6-base sequence is a "six-cutter" 21

Type II Restriction Enzymes No ATP requirement Recognition sites in dsDNA usually have a 2-fold axis of symmetry Cleavage can leave staggered or "sticky" ends or can produce "blunt” ends 22

Type II Restriction Enzymes Names use 3-letter italicized code: 1st letter - genus; 2nd,3rd - species Following letter denotes strain EcoRI is the first restriction enzyme found in the R strain of E. coli 23

Nucleic Acids Replication of DNA NEED ARTWORK FROM BRUICE BOOK

Nucleic Acids Enzymes break hydrogen bonds between DNA strands Replication of DNA T … A A … T C … G C … G A … T G … C A … T … C G C … G T … A NEED ARTWORK FROM BRUICE BOOK Enzymes break hydrogen bonds between DNA strands

Nucleic Acids Replication of DNA Parent strand Parent strand T A C G … C G A T NEED ARTWORK FROM BRUICE BOOK New complementary nucleotides are enzymatically introduced to the separating strands

Nucleic Acids Two new complementary daughter strands are produced Replication of DNA Parent strand Parent strand T … A A … T C … G C … G A … T G C A T C G G C C G G C NEED ARTWORK FROM BRUICE BOOK T A A T Daughter strands Two new complementary daughter strands are produced

Nucleic Acids Replication is complete, two identical DNA molecules Replication of DNA Parent strands … T A C G … T A C G NEED ARTWORK FROM BRUICE BOOK Daughter strands Replication is complete, two identical DNA molecules

Nucleic Acids RNA Messenger RNA (mRNA) – Carries genetic information from DNA to ribosomes. Ribosomal RNA (rRNA) – Make up the ribosomes where protein synthesis occurs. Transfer RNA (tRNA) – Transports amino acids to the ribosomes to make protein. NEED SOMETHING HERE

Nucleic Acids Transcription DNA Coding Strand 5′ 3′ 3′ 5′ DNA Template Strand NEED SOMETHING HERE

Nucleic Acids Transcription DNA Coding Strand 5′ 3′ 3′ 5′ DNA Template Strand NEED SOMETHING HERE Enzymes open up hydrogen bonds between DNA coding and template strands

Nucleic Acids mRNA is synthesized using DNA template Transcription DNA Coding Strand 5′ 3′ 3′ 5′ DNA Template Strand NEED SOMETHING HERE 5′ 3′ New mRNA Strand mRNA is synthesized using DNA template

Nucleic Acids Protein Synthesis – Translation Ribosomes are the site of translation, they are made up of rRNA and protein mRNA goes to ribosome Ribonucleotide sequences, codons, on the mRNA tell the ribosome what amino acids are needed in the new protein NEED SOMETHING HERE mRNA Strand Codons

Nucleic Acids Arg Ile Ala Phe Protein Synthesis – Translation Ribosome reads message from codon NEED SOMETHING HERE Arg Ile Ala Phe mRNA Strand Codons

Nucleic Acids Ile U–A–U Protein Synthesis – Translation tRNAs bring amino acids Amino acid is attached to the 3′ end of the tRNA Ile U–A–U NEED SOMETHING HERE Anticodon

Nucleic Acids Polymerase Chain Reaction (PCR) PCR is used to obtain large quantities of a known sequence of DNA In just a few hours PCR can amplify 105 molecules of DNA to 1011 molecules The key enzyme in PCR, Taq, is isolated from the thermophilic bacterium Thermus aquaticus (found in a hot spring in Yellowstone National Park) NEED SOMETHING HERE

Nucleic Acids Polymerase Chain Reaction (PCR) Target DNA Heat Denatures DNA, separating the strands 95°C NEED SOMETHING HERE

Primers are added, Taq needs double-stranded DNA to begin Nucleic Acids Polymerase Chain Reaction (PCR) Primers are added, Taq needs double-stranded DNA to begin NEED SOMETHING HERE

Nucleic Acids Polymerase Chain Reaction (PCR) Primers are added, Taq needs double-stranded DNA to begin Primers are annealed at 50°C NEED SOMETHING HERE

Taq polymerase Mg2+ and nucleotides Nucleic Acids Polymerase Chain Reaction (PCR) Taq polymerase Mg2+ and nucleotides NEED SOMETHING HERE

Nucleic Acids Polymerase Chain Reaction (PCR) Repeat Cycle NEED SOMETHING HERE Repeat Cycle Repeat Cycle