Nucleotides Flashcards
Metabolism of Xenobiotics: Phase 1 Reaction
Hydroxylation reactions
Enzyme: monooxygenases of cytochrome P450s
Medical compounds that are foreign to the body
Xenobiotics
Metabolism of Xenobiotics: Phase 2 Reaction
Conjugation reactions
Enzymes: Glucoronosyltransferases, sulfotransferases, glutathione S-transferases
The monomer units or building blocks of nucleic acids
Nucleotides
Nitrogen-containing heterocycles, cyclic compounds whose rings contain both carbon and other elements
Purines and Pyrimidines
Sugar + Purine or Pyrimidine; the link is a ring nitrogen
Nucleoside
Nucleosides with a phosphoryl group esterified to a hydroxyl group of the sugar
Nucleotides
5’-phosphoryl group forms a phosphodiester bond with the 3’-OH of another nucleotide; Pgosphodiesterases catalyze the hydrolysis of phosphodiester bonds
Polynucleotides
Purine ring is constructed by adding carbons and nitrogens to a preformed ribose-5-phosphate
Purine Synthesis
Purine Synthesis: Sources of atoms
Aspartic acid Glycine Glutamine Carbon dioxide N10-formyltetrahydrofolate and N5, N10-methenyltetrahydrofolate
In contrast, the pyrimidine ring is synthesized before being attached to ribose 5-phosphate
Pyrimidine Synthesis
Pyrimidine Synthesis: Sources of atoms
Glutamine
Aspartic acid
Carbon dioxide
What are the 2 compounds used in Purine Synthesis but not used in Pyrimidine Synthesis?
Glycine
N10-formyltetrahydrofolate and N5, N10-methenyltetrahydrofolate
An activated pentose that participates in the synthesis of purines and pyrimidines, and in the salvage of purine bases
Synthesis of 5-phosphoribosyl-1-pyrophosphate (PRPP)
Substrates: ATP and ribose 5-phosphate
Enzyme: PRPP synthetase
This is a committed step in purine nucleotide biosynthesis
Enzyme: glutamyl PRPP amidotransferase
Synthesis of 5’-phosphoribosylamine
9 steps that lead to the synthesis of IMP; “Parent” purine nucleotide
Synthesis of inosine monophosphate
Requires a two-step energy-requiring pathway; AMP synthesis requires GTP, while GMP synthesis requires ATP
Conversion of IMP to AMP and GMP
Purines that result from the normal turnover of cellular nucleic acids or that are obtained from the diet and not degraded, can be reconverted into nucleoside triphosphates and used by the body
Salvage Pathways for Purines
Salvage Pathways for Purines: Irreversible Enzymes
Adenine phosphoribosyltransferase (APRT) Hypoxanthine-guanine phosphoribosyltransferase (HGPRT)
Steps in De Novo Pyrimidine Synthesis: Regulated and Rate limiting step
Enzyme: Carbamoyl phosphate synthetase II
Inhibited by UTP
Activated by ATP and PRPP
Synthesis of Carbamoyl phosphate
Steps in De Novo Pyrimidine Synthesis: Dihydroorotate reductase is located inside the mitochondria; All the rest are cystosolic
Synthesis of Orotic Acid
Steps in De Novo Pyrimidine Synthesis: The “parent” pyrimidine nucleotide is Orotidine monophosphate (OMP)
Formation of Pyrimidine nucleotide
Steps in De Novo Pyrimidine Synthesis: Enzyme: CTP synthetase
Synthesis of UTP and CTP
Steps in De Novo Pyrimidine Synthesis: Enzyme: Thymidylate synthase; N5N10-methyltetrahydrofolate is the source of the methyl group
Synthesis of dTMP from dUMP
Few pyrimidine bases are salvaged in human cells
Salvage Pathway for Pyrimidines
Nucleoside diphosphates are synthesized from the corresponding nucleoside monophosphates using base-specific nucleoside monophosphate kinases
Conversion of nucleoside monophosphate to nucleoside diphosphates and triphosphates
Enzyme: Ribonucleotide reductase;
It is multisubunit enzyme that is specific for the reduction of necleoside diphosphates to their deoxy forms
Synthesis of Deoxyribonucleosides
Degradation of dietary nucleic acids occurs in the small intestines where a family of pancreatic enzymes hydrolyze the nucleotides to nucleosides and free bases
Purine Degradation
The pyrimidine ring can be opened and degraded to highly soluble structures
Pyrimidine Degradation
Sulfonamides are structural analogs of PABA that competitively inhibit bacterial synthesis of folic acid
PABA analogs
Methotrexate and TMP inhibit the reduction of dihydrofolate to tetrahydrofolate, catalyzed by dihydrofolate reductase
Folic Acid analogs
Hyperuricemia with recurrent attacks of acute arthritis caused by deposition of uric acid crystals
Gouty Arthritis
X-linked recessive deficiency in HGPRT that causes a rise in intracellular PRPP and hyperuricemia; triad of hyperuricemia, mental retardation, self-mutilation
Lesch-Nyhan Syndrome
Purine overproduction and hyperuricemia occurs secondary to enhanced generation of PRPP precursor ribose 5-phosphate
Von Gierke’s Disease
Leads to severe combined immunodeficiency (both T and B lymphocytes affected)
Adenosine deaminase deficiency
Metabolically converted to 5-FdUMP which becomes permanently bound to the inactivated thymidylate synthase
5-Fluorouracil
Low activities of orotidine phosphate decarboxylase and orotate phosphoribosyltransferase result in: Abnormal growth, megaloblastic anemia, excretion of large amounts of orotate in urine
Orotic Aciduria
Deoxyribonucleic acid; A polymer composed of nucleotide building blocks
DNA
5’-OH group attached to 3’-OH group; strands have directionality; bonds are cleaved hydrolytically by chemicals or hydrolyzed enzymatically by exonucleases or endonucleases
3’-5’ Phosphodieters bonds
Strands run in opposite directions
Antiparallel Strands
Held together by hydrogen bonds and hydrophobic interactions
Complementary base pairing
In any sample of dsDNA, the amount of adenine equals the amount of thymine, the amount of guanine equals the amount of cytosine
Chargaff’s Rules
Temperature at which one half of the helical structure is lost;
Under appropriate conditions, denaturation (annealing) may occur
Melting Temparature
Most common; Right-handed helix with 10 residues per 360 turn of the helix
B-DNA
Moderately dehydrated B form, also right-handed with about 11 base pairs per turn
A-DNA
Left-handed helix that contains about 12 base pairs per turn, naturally in regions of alternating purines and pyrimidines
Z-DNA
Five classes of small, positively charged proteins that form ionic bonds with negatively charged DNA
Histones
Further packing of DNA due to hydrophobic interactions and in association with other non-histone proteins compacts it into _____
Chromatin
Densely packed and transcriptionally inactive chromatin during interphase, observe by electron microscopy
Heterochromatin
Transcriptionally active chromatin that stains less densely
Euchromatin
Also called a nucleofilament; nucleosomes that are packed more tightly; Organized into loops that are anchored by a nuclear scaffold containing several proteins
Polynucleosome
Prokaryotic DNA Synthesis: Group of proteins that recognize the origin of replication
Step 1: DNA A protein
Prokaryotic DNA Synthesis: unwind the double helix ahead of the advancing replication fork
Step 2: Helicase
Prokaryotic DNA Synthesis: Maintain the separation of the parental strands
Step 3: Single-stranded DNA-binding proteins
Prokaryotic DNA Synthesis: Remove supercoils that interfere with the further unwinding of the double helix
Step 4: DNA topoisomerases
DNA Toposiomerase: Type I
Swivelase (cleaves one strand)
DNA Toposiomerase: Type II
Gyrase (cleaves both strands; target of quinolone antibiotics)
Prokaryotic DNA Synthesis: Synthesize short stretches of RNA called primers, needed by DNA polymerase to begin DNA chain elongation
Step 5: Primase
Prokaryotic DNA Synthesis: Catalyzes chain elongation, using 5’-deoxyribonucleoside triphosphates as substrates
Step 6: DNA Polymerase III
Prokaryotic DNA Synthesis: Removes RNA primers using its 5’-3’ exonuclease activity and fills in the resulting gaps
Step 7: DNA Polymerase I
Prokaryotic DNA Synthesis: Seals the nicks between Okazaki fragments and catalyzes the final phospholipid ester linkage
Step 8: Ligase
Stretches of highly repetitive DNA found at the ends of linear chromosomes; As cells divide and age, these sequences are shortened, contributing to cell death
Telomeres
Make a DNA copy of their RNA, integrate that copy into host cells; lack of proof reading explains high mutation rate
Reverse transcriptase
Mismatched strand, escaped proofreading
DNA Damage
Identification of the mismatched strand
DNA Repair
DNA Repair: Copying errors (single base or two to five base unpaired loops); Methyl-directed strand cutting, exonuclease digestion, and replacement
Mismatch Repair
DNA Repair: Spontaneous, chemical, or radiation damage to a single base; Base removal by N-glycosylase, abasic sugar removal, replacement
Base Excision Repair
DNA Repair: Spontaneous, chemical, or radiation damage to a DNA segment; Removal of an approximately 30-nucleotide oligomer and replacement
Nucleoside Excision Repair
DNA Repair: Ionizing radiation, chemotherapy, oxidative free radicals; Synapsis, unwinding, alignment, ligation
Double-Strand Break Repair
Polymers of nucleotides, but differ from DNA by containing: Ribose instead of deoxyribose, Uracil instead of thymine
RNA
“Rampant” because it is the most common type of RNA; Associated with several proteins as a component of the ribosomes
Ribosomal RNA or rRNA
“Tiny” because it is the smallest RNA; Adaptor molecule that carries a specific amino acid to the site of protein synthesis
Transfer RNA or tRNA
“Massive”; Carries genetic information from the nuclear DNA to the cytosol, where it is used as the template for protein synthesis
Messenger RNA or mRNA
A subset of RNAs significantly involved in mRNA processing and gene regulation
Small nuclear RNA or snRNA
4-subunit enzyme that synthesizes RNA; Possesses 5’-3’ polymerase activity
RNA polymerase
Recognizes the nucleotide sequence (promoter region) at the beginning of the length of the DNA to be transcribed
Sigma factor
Required for termination of transcription of some genes
Rho factor
Prokaryotic DNA Transcription: RNA polymerase holoenzyme binds to the promoter region
Step 1: Initiation
Prokaryotic DNA Transcription: RNA polymerase copying one strand of the DNA double helix, pairing Cs with Gs and As with Us
Step 2: Elongation
Prokaryotic DNA Transcription: Maybe accomplished by RNA polymerase alone or may require ρ factor
Step 3: Termination
Stretch of 6 nucleotides (5’-TATAAT-3’) centered about 8 to 10 nucleotides to the left of the transcription start site
Pribnow Box
Second consensus nucleotide sequence (5’-TTGACA-3’) about 35 bases to the left of the transcription start site
-35 Sequence
Classes of RNA polymerase: For large rRNAs in the nucleolus
RNA Polymerase I
Classes of RNA polymerase: For mRNAs
RNA Polymerase II
Classes of RNA polymerase: For tRNAs and some other small rRNAs in the nucleoplasm
RNA Polymerase III
TATA or Hogness box, CAAT box and GC box; Serve as binding sites for proteins called general transcription factors
Promoter Sequences
DNA sequences that increase the rate of initiation of transcription by binding to specific transcription factors called activators
Enhancers
Linear copy of the transcriptional unit, the segment of DNA between specific initiation and termination sequences
Primary transcript
Synthesized from long precursor molecules called preribosomal RNAs
rRNAs
Also made from longer precursor molecules; these must have an intervening sequence removed and the 5’ and 3’ ends of the molecule are trimmed by ribonuclease
tRNAs
Regulation of gene expression in prokaryotes usually involves either initiation or termination of transcription
Genetic Regulation
A set of structural genes coding for a group of proteins required for a particular metabolic function along with the regulatory region that controls the expression of the structural genes
Operon
Portion of the bacterial chromosome that controls the synthesis of the enzymes involved in lactose metabolism
Lactose Operon
Encodes a β-galactosidase
Z gene
Encodes a galactosidase permease, the transport protein required for the entry of lactose into the cell
Y gene
Encodes a lac repressor protein that is constitutively expressed and located at a distant site in the DNA
i gene
Encodes a thiogalactoside transacetylase enzyme, whose function is unknown
A gene
Proteins translated on Ribosomes associated with RER
Secreted proteins
Proteins inserted into the cell membrane
Lysosomal enzymes
Proteins translated on free cytoplasmic ribosomes
Cytoplasmic proteins
Mitochondrial proteins
Consists of three bases (triplet)
Codon
Total number of codons
64 codons
Total codons that code for amino acids
61 codons
Stop codons
Nonsense codons (UAA, UGA, UAG)
Start codon
Initiation codon (AUG)
A specific codon always codes from the same amino acid
Specific Genetic Code
It has been conserved from very early stages of evolution with only slight differences in the manner in which the code translated
Universal Genetic Code
A given amino acid may have more than one triplet coding for it
Redundant Genetic Code
Code is read from a fixed starting point as a continuous sequence of bases, taken three at a time
Commaless Genetic Code
Accurate base pairing is required only in the first 2 nucleotide positions of an mRNA codon, so codon differing in the 3rd wobble position may code for the same tRNA/amino acid
tRNA wobble
Binds an incoming aminoacyl-tRNA
A site codon
Occupied by peptidyl-tRNA
P site codon
Occupied by the empty tRNA as it is about to exit the ribosome
E site codon
Amino-acetyl-tRNA synthetase (1perAA) uses an ATP scrutinizes an amino acid before and after it binds to tRNA
Charging
DNA Translation: Activated by GTP hydrolysis, initiation factors (eIFs) help assemble the 40s ribosomal subunit with the initiator tRNA and are released when the mRNA and the ribosomal unit assemble with the complex
Step 1: Initiation
DNA Translation: Aminoacyl-tRNA binds to A site; Elongation factors direct the binding of the appropriate tRNA to the codon in the empty A site
Step 2: Elongation
DNA Translation: Releasing factors are proteins that hydrolyze the peptidyl-tRNA bond when a stop codon occupies the A site
Step 3: Termination
Energy Requirements of Translation
1) tRNA aminoacylation (ATP➡️AMP)
2) Loading tRNA onto ribosome (GTP➡️GDP)
3) Translocation (GTP➡️GDP)
Post-translational Modification
1) Trimming excess amino acids
2) Phosphorylation
3) Glycosylation
4) Hydroxylation
5) Destruction by Ubiquitin
Any permanent heritable change in the DNA base sequence of an organism; Has the potential to change the base sequence of mRNA and the amino acid sequence of proteins
Mutation
Point Mutation: Purine-Pyrimidine to Purine-Pyrimidine
Transition
Point mutation: Purine-Pyrimidine to Pyrimidine-Purine
Transversion
New codon specifies same amino acid, often base change in 3rd position of codon
Silent Mutation
New codon specifies a different amino acid
Missense Mutation
New codon is a stop codon; Shorter than normal protein, usually nonfunctional
Nonsense Mutation
Deletion or addition of a base; Protein usually nonfunctional, often shorter than normal
Frame shift Mutation
Unequal crossover in meiosis; Loss of function, protein shorter than normal or entirely missing
Large segment deletion Mutation
A splice site is lost through mutation
Splice donor or acceptor Mutation
Expansions in coding regions cause protein product to be longer than normal and unstable
Triple repeat expansion Mutation
Used to deduce original sequence of DNA; Dideoxynucleotides halt DNA polymerization at each base, generating sequences of various lengths that encompass the entire sequence
Sanger DNA Sequencing
Molecular biology lab procedure that is used to synthesize many copies of a desired fragment of DNA
Polymerase Chain Reaction
A DNA sample is electrophoresed on a gel and then transferred to a filter
Southern Blot
Similar technique but involves radioactive DNA probe binding to sample RNA
Northern Blot
Sample protein is separated via gel electrophoresis and transferred to a filter
Western Blot
Thousands of nucleic acid sequences are arranged in grids on glass or silicon
Microarrays
Enzyme-linked immunosorbent assay; A rapid immunologic technique testing for antigen-antibody reactivity
ELISA
Flourescence in situ hybridization; Flourescence probe binds to specific gene site of interest
FISH
Inherited difference in the pattern of restriction; Important in understanding various single-gene and multigenic diseases
Restriction Fragment Length Polymorphism
The production of recombinant DNA molecule that is self-perpetuating
Cloning
Treatment option for diseases caused by deficiency of a gene product
Gene Treatment
