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Lecture 21—BCH 4053—Summer 2000 Slide 1 Nucleotide Functions • Building blocks of nucleic acids • Triphosphates are energy intermediates • ATP major energy currency • GTP involved in driving protein synthesis • “Carriers” of metabolic intermediates • UDP intermediates in sugar metabolism • CDP intermediates in lipid metabolism • NAD and CoA are ADP intermediates • Chemical signaling “second messengers” • cyclic AMP and cyclic GMP Slide 2 Nucleic Acid Structure • Linear polymer of nucleotides • Phosphodiester linkage between 3’ and 5’ positions • See Figure 11.17 Slide 3 Nucleic Acid Sequence Abbreviations • Sequence normally written in 5’-3’ direction, for example: Guanine Adenine Cytosine H N O H2N H 2N N N N N N N H H O H O H O O O O O- O H H H O O P P P O- H H H O O H O P O H H H H H O H H H H O O N N O H N O N O Thymine NH2 N O- O O- Lecture 21, page 1 Slide 4 Sequence Abbreviations, con’t. Let Le tter sta nd for base: A G C T 5'-end P 3'e nd P P P P Let Letter stand for nucleoside 5'-end pApGpCpTp 3'-end Let Letter stand for nucleotide 5'-end AGCT 3'-end Slide 5 Biological Roles of Nucleic Acids • DNA carries genetic information • 1 copy (haploid) or 2 copies (diploid) per cell • See “History of Search for Genetic Material” • RNA at least four types and functions • • • • messenger RNA—structural gene information transfer RNA—translation “dictionary” ribosomal RNA—translation “factory” small nuclear RNA—RNA processing Slide 6 DNA Structure • Watson-Crick Double Helix • Clues from Chargaff’s Rules • A=T, C=G, purines=pyrimidines • Helical dimensions from Franklin and Wilkins X-ray diffraction studies • Recognition of complementary base pairing possibility given correct tautomeric structure (See Figure 11.20) Lecture 21, page 2 Slide 7 Nature of DNA Helix • Antiparallel strands • Ribose phosphate chain on outside • Bases stacked in middle like stairs in a spiral staircase • Figure 11.19—schematic representation • Complementary strands provide possible mechanism for replication • Figure 11.12 representation of replication process Slide 8 Size of DNA Molecules • 2 nm diameter, about 0.35 nm per base pair in length • Very long, millions of base pairs Organism • SV 40 virus • λ phage • E. coli • Yeast • Human Base Pairs MW Length 5.1 Kb 48 Kb 3.4x10 6 32 x 106 1.7 µm 17 µm 4,600 Kb 13,500 Kb 2.9 x 106 Kb 2.7 x 109 9 x 109 1.9 x 1012 1.6 mm 4.6 mm 0.99 m Slide 9 Packaging of DNA • Very compact and folded • E. coli DNA is 1.6 mm long, but the E. coli cell is only 0.002 mm long Histones are rich in the basic amino acids lysine and arginine, which have positive charges. These positively charged residues provide binding for the negatively charged ribose-phosphate chain of DNA. • See Figure 11.22 • Eukaryotic cells have DNA packaged in chromosomes, with DNA wrapped around an octameric complex of histone proteins • See Figure 11.23 Lecture 21, page 3 Slide 10 Messenger RNA • “Transcription” product of DNA • Carries sequence information for proteins • Prokaryote mRNA may code for multiple proteins • Eukaryote mRNA codes for single protein, but code (“exon”) might be separated by noncoding sequence (“introns”) • See Figure 11.24 Slide 11 Ribosomal RNA • “Scaffold” for proteins involved in protein synthesis • RNA has catalytic activity as the “peptidyl transferase” which forms the peptide bond • Prokaryotes and Eukaryotes have slightly different ribosomal structures (See Figure 11.25) • Ribosomal RNA contains some modified nucleosides (See Figure 11.26) Remember that the sedimentation rate is related to molecular weight, but is not directly proportional to it because it depends both on molecular weight (which influences the sedimentation force) and the shape of the molecule (which influences the frictional force). Slide 12 Transfer RNA • Small molecules—73-94 residues • Carries an amino acid for protein synthesis • One or more t-RNA’s for each amino acid • “Anti-codon” in t-RNA recognizes the nucleotide “code word” in m-RNA • 3’-Terminal sequence always CCA • Amino acid attached to 2’ or 3’ of 3’-terminal A • Many modified bases (Also Figure 11.26) Lecture 21, page 4 Slide 13 Small Nuclear RNA’s • Found in Eukaryotic cells, principally in the nucleus • Similar in size to t-RNA • Complexed with proteins in small nuclear ribonucleoprotein particles or snRNPs • Involved in processing Eukaryotic transcripts into m-RNA Slide 14 Chemical Differences Between DNA and RNA • Base Hydrolysis • DNA stable to base hydrolysis • RNA hydrolyzed by base because of the 2’-OH group. Mixture of 2’ and 3’ nucleotides produced • See Figure 11.29 • DNA more susceptible to mild (1 N) acid • Hydrolyzes purine glycosidic bond, forming apurinic acid Slide 15 Enzymatic Hydrolysis of Nucleic Acids • Many different kinds of nucleases in nature • Hydrolysis of phosphodiester bond • Exonucleases hydrolyze terminal nucleotides • Endonucleases hydrolyze in middle of chain. Some have specificity as to the base at which hydrolysis occurs Lecture 21, page 5 Slide 16 Enzymatic Hydrolysis, con’t. • Specificity as to the bond which is cleaved • a type cleaves the 3’ phosphate bond • Produces 5’-phosphate products • b type cleaves the 5’ phosphate bond • See Figure 11.30 • Examples: (Also see Table 11.4) • Snake venom phosphodiesterase • “a” specific exonuclease • Spleen phosphodiesterase • “b’ specific exonuclease • See Figure 11.31 Slide 17 Restriction Endonucleases • Enzymes of bacteria that hydrolyze “foreign” DNA • Name based on “restricted growth” of bacterial viruses • Enzymes specific for a short sequence of nucleotides (4-8 bases in length) • Methylation of the same sequence protects “self” DNA from hydrolysis Lecture 21, page 6