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Outline

Nitrogenous Bases The Pentoses of Nucleic acids Nucleosides are Formed by Joining a Nitrogenous Base to a Sugar Nucleotides - Nucleoside Phosphates Nucleic Acids are Polynucleotides Classes of Nucleic Acids Hydrolysis of Nucleic Acids

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.

Nitrogenous Bases
Know the basic structures Pyrimidines
Cytosine (DNA, RNA) Uracil (RNA) Thymine (DNA)

Purines
Adenine (DNA, RNA) Guanine (DNA, RNA)

Properties of Pyrimidines and Purines


Keto-enol tautomerism Acid/base dissociations Strong absorbance of UV light

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

Nucleosides

Linkage of a base to a sugar Base is linked via a glycosidic bond 1C links to 9 N of purine and to the 1 N of pyrimidine base Named by adding -idine to the root name of a pyrimidine or -osine to the root name of a purine Conformation can be syn or anti

Nucleotides
Nucleoside phosphates Know the nomenclature "Nucleotide phosphate" is redundant! Nucleotides are polyprotic acids

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

Nucleic Acids Polynucleotides


Polymers linked 3' to 5' by phosphodiester bridges Ribonucleic acid and deoxyribonucleic acid Know the shorthand notations Sequence is always read 5' to 3' In terms of genetic information, this corresponds to "N to C" in proteins

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

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

The Structure of DNA



An antiparallel double helix Diameter of 2 nm 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 Base pairs: A-T, G-C

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

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

Ribosomal RNA
Ribosomes are about 2/3 RNA, 1/3 protein rRNA serves as a scaffold for ribosomal proteins

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

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

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

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

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

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

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

Outline
Primary Structure of Nucleic Acids ABZs of DNA Secondary Structure Denaturation and Renaturation of DNA Tertiary Structure of DNA Chromosome Structure Chemical Synthesis of Nucleic Acids Secondary and Tertiary Structure of RNA

Primary Structure
Sequencing Nucleic Acids Chain termination method (dideoxy method), developed by F. Sanger Base-specific chemical cleavage, developed by Maxam and Gilbert Both use autoradiography - X-ray film develops in response to presence of radioactive isotopes in nucleic acid molecules

DNA Replication
DNA is a double-helical molecule Each strand of the helix must be copied in complementary fashion by DNA polymerase Each strand is a template for copying DNA polymerase requires template and primer Primer: an oligonucleotide that pairs with the end of the template molecule to form dsDNA DNA polymerases add nucleotides in 5'-3' direction

Chain Termination Method


Based on DNA polymerase reaction Run four separate reactions Each reaction mixture contains dATP, dGTP, dCTP and dTTP, one of which is P-32-labelled Each reaction also contains a small amount of one dideoxynucleotide: either ddATP, ddGTP, ddCTP or ddTTP

Chain Termination Method


Most of the time, the polymerase uses normal nucleotides and DNA molecules grow normally Occasionally, the polymerase uses a dideoxynucleotide, which adds to the chain and then prevents further growth in that molecule Random insertion of dd-nucleotides leaves (optimally) at least a few chains terminated at every occurrence of a given nucleotide

Chain Termination Method


Run each reaction mixture on electrophoresis gel Short fragments go to bottom, long fragments on top Read the "sequence" from bottom of gel to top Convert this "sequence" to the complementary sequence Now read from the other end and you have the sequence you wanted - read 5' to 3'

Chemical Cleavage Method


Not used as frequently as Sanger's Start with ssDNA labelled with P-32 at one end Strand is cleaved by chemical reagents Assumption is that strands of all possible lengths, each cleaved at just one of the occurrences of a given base, will be produced. Fragments are electrophoresed and sequence is read

Chemical Cleavage Method

Four reactions are used G-specific cleavage with dimethyl sulfate, followed by strand scission with piperidine G/A cleavage: depurination with mild acid, followed by piperidine C/T cleavage: ring hydrolysis by hydrazine, followed by piperidine C cleavage: same method (hydrazine and piperidine), but high salt protects T residues

Chemical Cleavage Method

Reading the gels... It depends on which end of the ssDNA was radioactively labelled! If the 5'-end was labelled, read the sequence from bottom of gel to top (5' to 3') If the 3'-end was labelled, read the sequence from top of gel to bottom (5' to 3') Note that the nucleotide closest to the P-32 will be missed in this procedure

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