Biochem Exam #1

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

-DNA is destroyed by DNAse not RNAse or proteases

H2A/H2B/H3/H4 x 2

-Octamer

-Nucleosome + Linker DNA + H1 Histones

-DNA + associated nucleoproteins

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

• -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 5%
• -tRNA 10%
• -rRNA 85%

• -Nucleus: splices hnRNA
• -Cytosol: targets proteins

• -Hydrogen bonding
• -Hydrophobic stacking

RNA

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

• -Strand separation
• -DNA polymerases

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

• -Polymerase Selectivity
• -Proofreading
• -Mismatch Repair

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

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

• -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)

• *1-2000 nt prokaryotes
• *~200 nt eukaryotes

-Requires terminal phosphate group

• -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)

• -Antibiotics
• -Inhibit DNA gyrase but not eukaryotic topo II

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

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

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

• -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

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

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

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

• -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

-Non-coding strand

-Coding strand

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

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

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

• -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)

-3 types

• -Type I (nucleolus)
• -Type II (mRNA)
• -Type III (tRNA)

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

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

Makes several proteins vs. One protein

• -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

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

-100-200 residues

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

• -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

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

• -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

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

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

• -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

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

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

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

• -AUG
• -5'-->3'
• -First tRNA enters P site

• -GTP hydrolyzed
• -Initiation factors required

-mRNA binds to small ribosomal subunit

• -5' cap recognition
• -Scans to first AUG (P site)
• -Methionine (First AA)
• -Greater than 12 eIFs

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

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

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

• -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

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

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

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

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

When transcribed

Always transcribed

Modulated expression (up or down)

• -Lac operon
• -Tryptophan attenuator

-A coordinated unit of gene expression

-Consists of: Regulator genes, Operator sites, Structural genes

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

-Lac repressor binds to operator site and prevents transcription

-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

• -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

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

• -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

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

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

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

No

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

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

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

• -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

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

-Repressor proteins have deacetylase activity

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

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

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

• -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)

• -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)

• -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

• -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

• -Depurination
• -Oxidation/Free Radical
• -Errors in Replication

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