biochemistry molecular biology high yield

Biochemistry Molecular Biology Part I Megan Murray M.D., Ph.D Candidate University of Buffalo BC01_1- Nucleic Acid Components • DNA and RNA are assembled from nucleotides: Nitrogenous base Pentose Phosphate • • Pentoses: Ribose → RNA; Deoxyribose → DNA Nitrogenous bases: Purines: rings Pyrimidines: ring Pure As Gold •FA 2013: 64.2 • FA 2012: 68.2 • FA 2011: 67.1 • ME 3e: 60.3 • ME 4e: 60.3 CUT the pie BC01_1- BC01_1- Nucleic Acid Components • • • Nucleoside: nitrogenous base + ribose Nucleotide: nucleoside + phosphate group(s) Nucleic acids: polymers of nucleoside monophosphates Structural elements of the most common nucleotides Commons.wikimedia.org Used with permission •FA 2013: 64.2 • FA 2012: 68.2 • FA 2011: 67.1 • ME 3e: 60.3 • ME 4e: 60.3 BC01_1- BC01_1- Nucleic Acid Composition • Phosphate group links 3’ carbon of deoxyribose to 5’ carbon of another deoxyribose • Each strand has 5’ and 3’ ends • Eukaryotes DNA is double-stranded RNA is single-stranded • Strands are joined by hydrogen bonds between bases • Bases of adjoining strands are always complementary A : T, C : G (DNA) U : T, C : G (RNA) Total purines = pyrimidines • Strands are anti-parallel 5’ to 3’ •FA 2013: 64.2 • FA 2012: 68.2 • FA 2011: 67.1 • ME 3e: 60.3 • ME 4e: 60.3 BC01_1- BC01_1- Chromosome Composition • Nuclear DNA is first packaged in a nucleosome Nucleosome • Negatively charged DNA winds around positively charged histone octamer • Histone octamer: copies of H2A, H2B, H3, and H4 • Histone H1: between adjacent nucleosomes • Difference between 10nm and 30nm chromatin is presence or absence of histone H1 •FA 2013: 64.1 • FA 2012: 68.1 • FA 2011: 66.1 • ME 3e: 60.3 • ME 4e: 60.3 BC01_1- BC01_1- Chromosome Composition Euchromatin • Loosely condensed nucleosomes • Accessible for transcription Heterochromatin • Densely condensed nucleosomes • Inaccessible for transcription •FA 2013: 64.1 • FA 2012: 68.2 • FA 2011: 66.1 • ME 3e: 60.3 • ME 4e: 60.3 BC01_1- BC01_1- DNA Replication Definition • Process by which genetic information is transmitted from cell to cell Process • complementary strands of parental DNA are pulled apart • Each is used as template for synthesis of new strand • Bidirectional and semi-conservative •FA 2013: 68.1 • FA 2012: 72.1 • FA 2011: 70.1 • ME 3e: 63.1 • ME 4e: 63.1 BC01_2- BC01_2- DNA Replication Sequence of events 5’ 3’ Base sequence recognized at origin of replication 3’ 5’ Helicase Origin 5’ 3’ 3’ 5’ 3’ 5’ Leading strand 5’ 1 3’ 3’ Lagging strand 5’ 3’ 3’ 3’ 5’ 5’ Single-stranded DNA binding protein (SSB) binds to each strand Primase synthesizes RNA primer (5’→ 3’) at origin of each parental strand to be complementary and antiparallel to DNA template strands DNA polymerase (III in prokaryotes and α/δ in eukaryotes) begins DNA synthesis Leading strand: continuous and toward replication fork Lagging strand: Okasaki fragments, away from replication fork 5’ 5’ Helicase breaks hydrogen bonds holding base pairs together → unwinding begins Site of quinolones, etoposide action! 3’ RNAse H removes primers, DNA polymerase fills gaps DNA polymerase has capacity to proofread by possessing 3’-exonuclease activity DNA ligase seals nicks between Okasaki fragments Topoisomerase II (DNA gyrase) relieves strain of supercoiling Replication is completed when forks meet •FA 2013: 68.1 • FA 2012: 72.1 • FA 2011: 70.1 • ME 3e: 63.1 • ME 4e: 63.1 BC01_2- BC01_2- Telomerases Telomeres • Repetitive sequences at ends of linear DNA molecules • Replication shortens telomere length, contributing to genetic material loss and cell aging Telomerase • Enzyme which maintains telomere length (embryonic, germ, and stem cells) by replacing telomere sequences on parent strand • Inactive in somatic cells (skin, blood, connective tissue) • Prokaryotes have single circular chromosomes, hence no telomerases • Inappropriately present in cancer cells • FA 2013: NA • FA 2012: NA • FA 2011: NA • ME 3e: NA • ME 4e: NA BC01_2- BC01_2- DNA Repair 3’ Nucleotide excision repair T A 5’ T A 5’ 3’ UV • Excision endonuclease nicks the phosphodiester backbone of damaged strand 3’ • Removes defective oligonucleotide • DNA polymerase fills gap & DNA ligase seals nick 5’ T=T A 5’ A 3’ Excision endonuclease Xeroderma pigmentosum Xeroderma pigmentosum (XP) 3’ 3’ • Enzyme deficiency leads to XP 5’ T=T 5’ • Autosomal recessive 3’ A A 5’ • Extreme sensitivity to sunlight DNA polymerase Nick 3’ • Skin cancer (melanomas) 5’ T A • Diagnosis: excision endonuclease levels 5’ •FA 2013: 69.1 • FA 2012: 73.1 • FA 2011: 71.1 • ME 3e: 64.2 • ME 4e: 64.2 3’ DNA ligase • Skin freckling 5’ T A 3’ BC01_3- BC01_1- Homocysteinuria Signs and symptoms • ↑ homocysteine and methionine α -ketobutyrate • Marfanoid features + spontaneous thromboembolism Cysteine Cystathionine Cystathionine synthase CH3 THF B6 Homocysteine Homocysteine methyltransferase B12 Methionine S-Adenosylhomocysteine • Methyl groups for biosynthesis • Epinephrine • N-methylguanosine cap on mRNA ATP Pi + PPi • FA 2013: 109.1 • FA 2012: 113.1 • FA 2011: 108.3 • ME 3e: 48 • ME 4e: 48 S-Adenosylmethionine BC08_2-5 S-Adenosylmethionine Homocysteine methyl transferase N5-methyl THF B12 From diet Methionine Homocysteine S-Adenosylhomocysteine Methyl groups for biosynthesis of epinephrine N-methylguanine cap on mRNA ATP Pi + PPi • FA 2013: 109.1 • FA 2012: 113.1 • FA 2011: 108.3 • ME 3e: 47 • ME 4e: 47 S-Adenosylmethionine BC08_3-1 Folate Metabolism • FA 2013: 92.2 • FA 2012: 97.2 • FA 2011: 92.2 • ME 3e: 56 • ME 4e: 56 BC08_3-2 Catecholamine Synthesis Specific products derived from amino acids: • Tyrosine → thyroid hormones, melanin, catecholamines • Tryptophan → serotonin, NAD, NADP • Arginine → NO • Glutamate → GABA • Histidine → Histamine • Glycine → Heme • FA 2013: 108.1 • FA 2012: 112.1 • FA 2011: 107.1 • ME 3e: 46 • ME 4e: 46 BC08_3-3 Heme Synthesis • B6 deficiency → sideroblastic anemia • Iron deficiency → microcytic hypochromic anemia • Lead poisoning → microcytic sideroblastic anemia • FA 2013: 358.1 • FA 2012: 386.1 • FA 2011: 354.1 • ME 3e: 46 • ME 4e: 46 BC08_4-1 Vitamin B6 Deficiency vs Iron Deficiency vs Lead Poisoning Vitamin B6 Deficiency Iron Deficiency Lead Poisoning Microcytic Microcytic Microcytic; coarse basophilic stippling in erythrocytes Ringed sideroblasts in bone marrow - Ringed sideroblasts in bone marrow ↓ protoporphyrin ↑ protoporphyrin ↑ protoporphyrin ↓δ-ALA Normal δ-ALA ↑δ-ALA ↑ ferritin ↓ ferritin ↑ ferritin Normal serum iron ↓ serum iron ↑ serum iron Caused by isoniazid (TB) Caused by dietary iron insufficient to compensate for normal loss Caused by lead paint, pottery glaze, batteries • FA 2012: 386 • FA 2011: NA • FA 2010: NA • ME 3e: NA • ME 4e: NA • FA 2013: NA • FA 2012: 386 • FA 2011: NA • ME 3e: NA • ME 4e: NA BC08_4-2 Iron Metabolism Hemochromatosis • Mutation of HFE gene → ↑ iron absorption Ferroxidase (ceruloplasmin) • Ferritin binds iron → hemosiderin → deposits in tissues Liver cirrhosis and dermatitis Bronze skin • FA 2013: NA • FA 2012: NA • FA 2011: NA • ME 3e: NA • ME 4e: NA BC08_4-3 Heme Catabolism and Bilirubin UDP-glucuronyl transferase deficiency Kernicterus: accumulation of bilirubin in the brain (basal ganglia) • FA 2013: 358.1 • FA 2012: 386.1 • FA 2011: 354.1 • ME 3e: 46 • ME 4e: 46 BC08_4-4 Biochemistry Purines and Pyrimidines Megan Murray, M.D., Ph.D Candidate University of Buffalo Nucleotide Synthesis and Functions Nucleotide functions DNA and RNA synthesis Energy transport Nucleotide synthesis De novo synthesis (predominantly liver) Salvage pathway (peripheral cells outside the liver) • FA 2013: 65.1 • FA 2012: 69.1 • FA 2011: 67.1 • ME 3e: 60 • ME 4e: 60 BC09_1-2 Pyrimidine Synthesis • FA 2013: 65.1 • FA 2012: 69.1 • FA 2011: 67.1 • ME 3e: 61 • ME 4e: 61 BC09_2-1 Pyrimidine Synthesis: OTC Deficiency Ornithine transcarbamylase deficiency (OTC) • • • • Urea cycle Orothic aciduria Hyperammonemia No megaloblastic anemia Uridine monophosphate synthase deficiency • • • • Pyrimidine synthesis Orothic aciduria No hyperammonemia Megaloblastic anemia Pyrimidine Synthesis Pathway Urea Cycle • FA 2013: 65.2 • FA 2012: 69.2 • FA 2011: 67.2 • ME 3e: 61 • ME 4e: 61 BC09_2-2 De Novo Purine Synthesis Allopurinol 6-mercaptopurine • FA 2013: 65.1 • FA 2012: 69.1 • FA 2011: 67.1 • ME 3e: 62 • ME 4e: 62 BC09_3-1 Purine Salvage Pathway • FA 2013: 66.1 • FA 2012: 70.1 • FA 2011: 69.1 • ME 3e: 62 • ME 4e: 62 BC09_3-2 Purine Salvage Pathway Severe Combined Immunodeficiency • Lack of B- and T-cell function • Recurrent infections Allopurinol • Inhibits xanthine oxidase Hyperuricemia Adenosine deaminase deficiency • Autosomal recessive • Leads to accumulation of dATP • Orange, needle-shaped sodium urate crystals in diapers of infants • Crystal precipitation in joints induces inflammation gouty arthritis • Crystal precipitation in kidneys urate nephropathy • FA 2013: 66.1 • FA 2012: 70.1 • FA 2011: 69.1 • ME 3e: 62 • ME 4e: 62 BC09_3-3 ... 75.1 • FA 2010: 75.1 • ME 3e: 66.1 • ME 4e: 66.1 BC01_4- BC01_4- tRNA • tRNA carries activated (high energy) amino acid for translation • Each tRNA carries one activated amino acid determined... its anticodon sequence • Amino acid activation catalyzed by aminoacyl-tRNA synthetase Requires high energy bonds from ATP Transfers activated amino acid onto 3’ end of correct tRNA • tRNA has... Phase • Three-step cycle repeated for each amino acid added to protein after initiator Met • Four high energy bonds used per cycle: Two ATPs for charging tRNA and peptide bond formation Two GTPs