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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
Nitrogenous Bases
Know the basic structures Pyrimidines
Cytosine (DNA, RNA) Uracil (RNA) Thymine (DNA)
Purines
Adenine (DNA, RNA) Guanine (DNA, RNA)
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
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?
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
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
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
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
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
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
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