Biochem Exam #1

Griffith

-Genetic components are not killed by heat-Transformation and recombination can occur-Proved genetic component existed

Avery, MacLeod, McCarty

-DNA is destroyed by DNAse not RNAse or proteases

Nucleosome

H2A/H2B/H3/H4 x 2-Octamer

Solenoid

-Nucleosome + Linker DNA + H1 Histones

Chromatin

-DNA + associated nucleoproteins

Histones

-Small basic proteins (Lys, Arg), + charged

Junk DNA

-Regulates transcription/translation of protein coding sequences-Highly conserved-May be involved: chromosome structure/centromere function/homolog recognition-Evolution -Vital role for organisms to adapt to changes

mRNA, tRNA, rRNA %

-mRNA 5%-tRNA 10%-rRNA 85%

Small RNA

-Nucleus: splices hnRNA-Cytosol: targets proteins

How is the double helix stabilized?

-Hydrogen bonding-Hydrophobic stacking

2nd important nucleic acid in cells?

RNA

Qualities of DNA Replication

-Semiconservative (Meselson & Stahl)-DNA polymerase + host of other proteins-Accuracy & Speed-S phase (Eukaryotes)

DNA Replication Requires?

-Strand separation-DNA polymerases

Qualities of DNA Polymerases?

-5'-->3' Synthesis-High Fidelity-Accuracy, Speed

Contributions to Fidelity

-Polymerase Selectivity-Proofreading-Mismatch Repair

DNA Polymerase Activity

-5'-->3' polymerase activity-5'-->3' exonuclease, removal of primer-3'-->5' exonuclease, proofreading activity

DNA Initiation

-Bidirectional-Negative supercoiling allows binding of dnaA protein which unwinds/synthesizes primer-dnaB & dnaC: bind dnaA to bend/open the helix

RNA Primer for DNA Synthesis

-Required-Primase (RNA polymerase) synthesizes an RNA primer which is ~5 nt long-DNA polymerase III can now begin DNA synthesis-This primer is removed by DNA polymerase I (5'-->3' exonuclease activity)

Okazaki Fragments

*1-2000 nt prokaryotes*~200 nt eukaryotes

DNA Ligase

-Requires terminal phosphate group

Topoisomerases

-Unwind and wind DNA -Introduces transient (not permanent) strand breaks to relax DNA-Type I: single strand break-Type II: double strand break (known as DNA gyrase in prokaryotes, hydrolyzes ATP)

Ciprofloxacin & Novobiocin

-Antibiotics-Inhibit DNA gyrase but not eukaryotic topo II

Captothecin

-Inhibits topo type IB (eukaryotic topoisomerase) by stabilizing enzyme-DNA intermediate-Anticancer agent since nicked DNA can't replicate

DNA Replication in Eukaryotes

-Autonomous replication sequence (ARS)-8 Proteins, 11 Base Pair Consensus Sequence

Sliding Clamp Theory

-Asymmetrical-Processivity (Allows DNA pol to stay on DNA & not fall off)-Proofreading (3'-->5' exonuclease activity)

Telomeres

-Tandem Repeats-Hexanucleotide sequence-Species specific-TTAGGG for humans -On eukaryotes-Free ends of chromosomes present unique problem-3' end of lagging strand -Have telomerase (reverse transcriptase) which contains an RNA molecule & species specific

Telomerase in regards to Cancer

-Active in cancer cells-Keeps telomeres long-Drugs target telomerase by stopping tumor cell proliferation, thus shortening cancer cells telomeres

Telomerase in regards to Aging

-Telomeres shorten with time-Damaged skin, blood vessels, retinal cells can be prolonged by introducing telomerase

Qualities of Transcription

-Gene Expression-Regulate mRNA to regulate gene expression-On/off, Rate, Stability, Amount of specific transcripts

Transcription Initiation

-Sigma subunit (prokaryotes) of RNA polymerase recognizes a promoter region (no primer needed)-First phosphodiester bond formed-ATP or GTP (purine triphosphate) base pairs to template at +1 site-Consensus sequences: homologous sequence with same function -Prokaryotes: -35 sequence, Pribnow box

Template Strand

-Non-coding strand

Nontemplate

-Coding strand

Transcription Elongation

-Transcription bubble around 17 bases -RNA synthesized from template (noncoding strand) -No proofreading

Transcription Termination for Prokaryotes

-RNA hairpin followed by U-rich region (structural/metabolic genes)-Rho protein pulls RNA from template strand (ribosomal genes)

Transcription Termination for Eukaryotes

-No strong termination signal-100 nt downstream-Processing determines final 3' end

Bacterial RNA Polymerase

-1 RNA polymerase that transcribes all genes-Primase (specialized RNA polymerase which makes RNA primer needed in DNA synthesis, not involved in transcription)-Rifampicin (inhibits initiation of RNA synthesis)

Eukaryotes RNA Polymerase

-3 types-Type I (nucleolus)-Type II (mRNA)-Type III (tRNA)

RNA Polymerase Functions

-Scans for DNA initiation sites-Unwinds short stretches of DNA (17 bp)-Selects nucleotides-Catalyzes phosphodiester bond formation at +1-Interacts with regulatory proteins

Transcription in Eukaryotes

-Different promoters-Upstream regions have enhancers-Extensive processing of eukaryotic RNA

Polycistronic vs. Monocistronic

Makes several proteins vs. One protein

Eukaryotic Promoter Sequence

-Cis elements: promoter or enhancer sequences on DNA-CAAT box, Hogness (TATA) box: These sequences are recognized by RNA pol II-Trans elements: bind to cis elements

Processing of mRNA

-Methylated G nucleotide (5'-5' linkage)--> Cap-Introns are removed-Poly A tail-100-200 residues

Split" Genes

-Introns intervene between exons-Introns varies slightly-Exons range from 45-249-Accurate splicing required

RNA Splicing

-Spliceosome: primary transcript + snRNP's-Small nuclear ribonucleoproteins (snRNPs): proteins + small RNA's (snRNA)-Lariet: excised intron-siRNA: small inhibitory RNAs-transcribed from some of that junk DNA areas

Actinomycin D

-Antibiotic-Inhibits RNA synthesis-Intercalates in dsDNA, prevents DNA from serving as a template-Works in bacteria and eukaryotes

Alpha-Amanitin

-Toxin in poisonous mushrooms-Inhibits Eukaryotic RNA polymerase-Type I (nucleolus) --> insensitive, can't block-Type II (mRNA) --> very sensitive, strongly inhibited-Type III (tRNA) --> inhibited only at high concentration

Rifamycin & Rifampin

-Changes RNA pol conformation so it can't initiate RNA synthesis-Doesn't bind to eukaryotic polymerase, only bacteria polymerase-Used to treat tuberculosis

Ribosomes in Prokaryotes and Eukaryotes

-Eukaryotes have larger ribosomes-Both eukaryotes and prokaryotes have large and small subunit

Ribosomal RNA

-Essential for protein synthesis-Cleavage in rRNA abolishes protein synthesis-P site sequence is conserved-rRNA needed for peptide bond formation-Antibiotics interact with rRNA

How is tRNA activated?

-Amino acid esters are activated intermediates in protein synthesis-2 ATP required to charge the tRNA -Aminoacyl tRNA synthetase: enzyme for each amino acid

Aminoacyl-tRNA Synthetase

-Increases fidelity of protein synthesis-Highly selective-Corrects own erros by hydrolyzing incorrect aminoacyl-adenylate

Codon Recognition

-AA not involved-Codon (mRNA) matches with Anticodon (tRNA)

Initiation of Protein Synthesis (Translation)

-AUG-5'-->3' -First tRNA enters P site-GTP hydrolyzed-Initiation factors required

Initiation of Translation (Eukaryotes)

-mRNA binds to small ribosomal subunit-5' cap recognition-Scans to first AUG (P site) -Methionine (First AA)-Greater than 12 eIFs

Initiation of Translation (Prokaryotes)

-Shine-Delgarno sequence (purine rich) indicates AUG start-Formyl-Met (First AA)-3 IFs

Peptidyl Transferase

-Catalyzes formation of peptide bond -Does so by nucleophilic attack-Energy from incoming AA-tRNA-Activity catalyzed by A residue of 23S rRNA

Translocase

-EF-G (Translocase) catalyzes the movement of peptidyl-tRNA out of the A site

Termination of Translation

-Release Factors read stop codons (UAA, UAG, UGA)-NO tRNA for stop codons-Release factors alter specificity of peptidyl transferase-Water used as an acceptor

Puromycin

-Prokaryotes & Eukaryotes-Analog of aminoacyl-tRNA -Premature chain termination

Erythromycin

-Prokaryotes-Binds irreversibly to large subunit (50S)-Inhibits translocation

Streptomycin

-Prokaryotes-Binds to small subunit (30S)-Inhibits initiation, causes misreading of mRNA

Diptheria Toxin

-Inhibits translocation-Blocks translocase by ADP-ribosylation of EF2 catalyzed by A-fragment of toxin

Gene is expressed

When transcribed

Constitutive expression

Always transcribed

Regulated expression

Modulated expression (up or down)

Regulation of Gene Transcription

-Lac operon-Tryptophan attenuator

Operon

-A coordinated unit of gene expression-Consists of: Regulator genes, Operator sites, Structural genes

Z, Y, A Gene

-Z gene: Beta galactosidase (Lactose--> Glucose + Galactose)-Ygene: Permease-A gene: Transacetylase

No Lactose

-Lac repressor binds to operator site and prevents transcription

Presence of Lactose

-The inducer binds to the repressor which prevents it from binding to the operator so the genes can be transcribed-When glucose is absent and lactose is present, the lactose with convert to 1,6 allolactose (inducer)-It binds to the repressor and inactivates the repressor-1,6 allolactose=Alpha-1,6 linked galactose + glucose-RNA polymerase binds to the promoter now and transcription occurs

Catabolite Repression in the Lac Operon

-When glucose concentration is low, cAMP increases-cAMP binds to CAP (Catabolite Activator Protein)/CRP (cAMP Response Protein)-This stabilizes the RNA polymerase and enhances transcription 50X

Lactose and Glucose Present

-Low cAMP levels -CAP doesn't bind polymerase-Little/no transcription of lac operon genes

Lactose but no Glucose

-Inducer binds repressor-Repressor falls off and becomes inactive -cAMP levels are high when glucose is not present-cAMP binds to CAP and stabilizes polymerase-Transcription occurs

Tryptophan Operon

-Encodes 5 enzymes that convert chorismate into tryptophan -Contains 14 AA peptide (with 2 Trp next to each other) + Untranslated attenuator sequence

High Trp

-High levels of Trp are available, ribosome passes quickly and termination turn forms-Transcription is blocked because there are high levels of Trp readily available

Low Trp

-Low levels of Trp causes the ribosomes to stall at Trp codons, the alternative turn forms, which prevents formation of termination turn-Transcription occurs

Post-transcriptional Regulation

-Other attenuate operons include:-Threonine, Phenylalanine, Histidine

Are genes organized in Operons?

No

Positional Information

-Cells can signal each other and modify gene expression depending on position

Histone Code Hypothesis

-Specific combinations of modifications help determine chromatin configuration & influence transcription-Chromatin remodeling

Ways to Alter Genes Available for Transcription

-Methylation of DNA (Deactivates it) -Gene Rearrangement (Ig)-Gene Amplification (Response to stimuli)

Heterochromatin vs. Euchromatin

-Heterchromatin: Condensed, little or no gene expression-Euchromatin: Open, transcriptionally active -Normal chromatin blocks TFIID & Pol II from associating with DNA-Transcriptionally active genes show DNase I hypersensitivity

Acetylation of Histones

-Transcription occurs-Lysine (+) residues on histones accept acetyl groups and decreases affinity of DNA to histones

Deacetylation of Histones

-Repressor proteins have deacetylase activity

Proteins Regulating Transcription (Trans-elements)

-General Transcription Factors (Initiation)-Activators & Repressors (Regulation)

Enhancers (Regulating Eukaryotic Gene Expression)

-Modulate activity of RNA polymerase (Bind Transcription Factors)-Position & orientation independent

Silencers (Regulating Eukaryotic Gene Expression)

-Control regions of DNA-Area where TF bind and represses by binding to repressors

General Transcription Factors (Basal Factors)

-Pre-initiation complex -Basal levels-TFIIA-TFIIH (Each have specific function, TATA & promoter recognition, Pol II & TF recruitment, Bind sequentially, 1-12 subunits, 15-250 kDa)

Homeotic (Hox) Genes

-Transcription factors that bind DNA-Position-specific differentiation & body segmentation during development-Wide variety of organisms-Homeobox: 180nt semiconserved sequences that codes for helix-turn-helix binding domain (homeodomain)

Steroid Hormones

-Transcription Factor-Derived from cholesterol-Alters PATTERN of expression rather than individual gene-Receptors bind to steroid hormones/retinoids/thyroid hormone -Conformation change allows binding of co-activator -Steroid binds to the hormone, causes conformational change & uncovers Zinc finger DNA binding domain-The complex interacts with GRE (regulatory DNA sequences)-The complex interacts with co-activator proteins and controls transcription of targeted genes

Post-transcriptional Regulation

-Alternative Splicing (Allows diversity, many proteins from a single gene is made)-mRNA Stability (Signal near 3' end, AU rich leads to degradation, especially in regulatory proteins, growth factors, transcription factors)-RNA Editing-Regulation of Translation

Endogenous Mutagenesis

-Depurination-Oxidation/Free Radical-Errors in Replication

Exogenous Mutagenesis

-Ionizing radiation resulting in free radicals-Alkylating-Nitrous acid=deamination-Ethidium bromide=intercalation-Ultraviolet