DIFFERENCES IN THESE REPEATS IS THE BASIS FOR DNA FINGERPRINTING
MICROSATELLITE REPEATS
POLYMORPHIC WITH 1-15 REPEATS.
DI, TRI, OR VNTR (VARIABLE # TANDEM REPEATS)
HUNTINGTON'S CHORIA (TRI CAG REPEAT >30)
INTERSPERSED REPETITIVE DNA
FOUND IN ALL EUK GENOMES
PROPAGATE BY RNA MEDIATED TRANSPOSITION
LINES
LONG INTERSPERSED NUCLEAR ELEMENTS
TRANSCRIBED INTO RNA USING RNA POL I PROMOTER LOCATED INSIDE GENE
CODE FOR ENZYME REVERSE TRANSCRIPTASE AND ENDONUCLEASE (RNase)
COPY THEMSELVES AND ENLARGE GENOME (21% IS ARE LINES)
SINES
SMALL INTERSPERSED NUCLEAR ELEMENTS
REVERSE TRANSCRIBED RNA ORIGINALLY TRANSCRIBED BY RNA POL III INTO tRNA, rRNA, and other small nuclear RNA
DO NOT CONTAIN ANY CODING SEQUENCES
31% OF GENOME
HIGHLY REPETITIVE SEQUENCE
REPEATED PATERN CAN BE 1 TO SEVERAL THOUSAND BP LONG AND IN TOTAL CAN BE SEVERAL MEGA BP
MOSTLY IN TELOMERIC AND CENTROMERIC REGIONS
3'UTR REGULATORY SEQUENCES
3'-UNTRANSLATED REGION
POLY-A SIGNAL FOR POST TRANSCRIPTIONAL MODIFICATION
BINDING SITES FOR PROTEINS
BINDING SITES FOR miRNA
tRNA COMPONENTS (6)
(1) 5'-TERMINAL PHOSPHATE GROUP
(2) ACCEPTOR STEM FOR ADDITION OF 3' CCA SEQUENCE
(3) CCA TAIL ON 3' END ADDED POST-TRANSCRIPTIONALLY. WHERE AMINO ACID ATTACHES
(4) D ARM. 4 BP LOOP OFTEN CONTAINING DIHYDRURIDINE
(5) ANTICODON ARM
(6) T ARM CONTAINING PSEUDO-URIDINE
EUK rRNA
28S, 18S, 5.8S, AND 5S + 82 PROTEINS
MAKE 80S RIBOSOMES (60S LARGE AND 40S SMALL)
60S (28S, 5.8S, 5S + 49 PROTEINS)
40S (18S + 33 PROTEINS)
80-85% OF TOTAL RNA FOUND IN AVERAGE EUK CELL
snRNA
SMALL NUCLEAR RNA FOUND WITHIN NUCLEUS OF EUK CELLS
TRANSCRIBED BY RNA POL II OR III
INVOLVED IN RNA SPLICING, REGULATION OF TRANSCRIPTION FACTORS, AND MAINTAINING TELOMERES
miRNA
microRNA POST-TRANSCRIPTIONAL REGULATORS THAT BIND TO COMPLEMENTARY SEQUENCES IN THE 3' UTR OF TARGET mRNA
USUALLY BLOCK TRANSLATION OR ACCELERATE DEGRADATION
CAN CAUSE METHYLATION OF GENES
TARGET ABOUT 60% OF MAMMALIAN GENES
FIRST STEP IN DNA REPLICATION
FORMATION OF PRE-INITIATION REPLICATION COMPLEX (pre-RC = ORC + MCM) REFERED TO AS LICENSING
Cdc6 LOADS MCM COMPLEX TO ORIGIN. MADE ONLY DURING G1 PHASE, PHOSPHORYLATED AT S PHASE AND DEACTIVATED.
MCM IS A DNA HELICASE
SSB PROTEINS
SINGLE STRANDED BINDING PROTEINS BIND TO DNA DURING SYNTHESIS TO KEEP FROM ANNEALING AND STABILIZE DNA TO ALLOW 100X FASTER REPLICATION
EUK LAGGING STRAND SYNTHESIS ENZYME
DNA POL ALPHA AND DELTA
DNA POL ALPHA ADDS RNA PRIMERS
PRIMERS REMOVED BY RNAse H & FLAP ENDONULEASES (FENs)
REPLACED BY DNA POL DELTA & LINKED BY DNA LIGASE
DNA POL ALPHA
SYNTHS AN RNA PRIMER AT INITIATION SITES FOR LEADING/LAGGING STRAND
PROVIDES 3'OH FOR ELONGATION WITH DNA NUCLEOTIDES
DNA POL BETA
IMPLICATED IN DNA BASE EXCISION REPAIR
DNA POL GAMMA
REPLICATES AND REPAIRS MITOCHONDRIAL DNA
HAS PROOFREADING 3'-5' EXONUCLEASE ACTIVITY
DNA POL DELTA
HIGHLY PROCESSIVE 5'-3' SYNTH
PROOFREADING 3'-5' EXONUCLEASE ACTIVITY
MAIN POL FOR DNA LEADING/LAGGING STRAND SYNTH AND GAP FILLING AFTER REMOVAL
CLAMP PROTEINS
CONTRIBUTE TO DNA SYNTH BY FORMING A SLIDING CLAMP AROUND DNA
HELPS RELEASE DNA POL
PCNA
PROLIFERATING CELL NUCLEAR ANTIGENS
PROTEIN THAT ACTS AS A PROCESSIVITY FACTOR FOR DNA POL DELTA IN EUK CELLS
TYPE OF DNA CLAMP
TELOMERASE
EXTENDS REPETITIVE SEQUENCES OF THE TELOMERE TO PREVENT DEGRADATION AND CHROM FUSION
REVERSE TRANSCRIPTASE THAT CARRIES OWN RNA MOLECULE, WHICH IS USED AS TEMPLATE DURING ELONGATION
REGULATED DURING DEVELOPMENT AND HAS VERY LOW ACTIVITY IN SOMATIC CELLS (AGING, CANCER)
PROK DNA REPLICATION
BIDIRECTIONAL
SINGLE ORIGIN
PROCARYOTIC DNA POLS
POL I -- DNA REPAIR, HAS 5'-3' ACTIVITY AND BOTH 3'-5' AND 5'-3' EXONUCLEASE ACTIVITY (REMOVING DNA PRIMERS) AND FILLS GAPS AFTER REMOVAL OF RNA PRIMERS
POL II -- REPAIR OF DAMAGED DNA. HAS 3'-5' EXONUCLEASE ACTIVITY
POL III -- MAIN POL IN PROC SYNTH. HAS 5'-3' SYNTH AND 3'-5' PROOFREADING
NOVOBIOXIN
ANTIBIOTIC
TARGET DNA GYRASE (TOPO I & II) AND BLOCK SYNTH
USED TO TREAT MRSA
NALADIXIC ACID
ANTIBIOTIC
EFFECTIVE AGAINST BOTH GRAM + & -
PREVENTS REANNEALING OF DOUBLE-SRTANDED BREAK
TREAT AGAINST URINARY TRACT INFECTIONS
CIPROFLOXIN
ANTIBIOTIC
ACTS ON TOPOs
TREATS ANTHRAX
REVERSE TRANSCRIPTASE
CAN TRANSCRIBE BOTH DNA AND RNA
RNase ACTIVITY REMOVES RNA FROM DNA/RNA HYBRIDS AND MAKES 2X STRANDED DNA
LACKS PROOFREADING & MAKES LOTS OF MISTAKES (1 IN 2,000 -- ABOUT 5X)
MATURE VIRUS PROTEINS
(GAG) GROUP SPECIFIC PROTEINS MAKES CORE AND STRUCTURAL PROTEINS (VIRAL CAPSID)
POLIMERASE CODES FOR:
--REVERSE TRANSCRIPTASE
--PROTEASE CLEAVES PROTEINS TO MAKE GAG AND POL
--INTEGRASE FOR INTEGRATION INTO HOST DNA
ENV CODES FOR ENVELOPE PROTEINS ESSENTIAL FOR VIRAL ENTRY INTO CELL
LTR LONG TERMINAL REPEATS
POL POLYMERASE GENE
ENV ENVELOPE GENE
TAT TRANSACTIVATOR PROTEIN
REV REGULATOR OF EXPRESSION
VIF VIRAL INFECTIVITY
VPU VIRAL PROTEIN U
VPR VIRAL PROTEIN R
NEF NEGATIVE-REGULATION PROTEIN
VIRAL RNA
DIMER RNA
5' CAP AND 3'POLLY A TAIL
TERMINAL NONCODING REGIONS IMPORTANT FOR REPLICATION AND INTERNAL REGIONS THAT ENCODE VIRAL PROTEINS
CAMPTOTHECIN
BINDS TO TOPO I AND DNA COMPLEX
PREVENTS RELIGATION
WORKS WITH CTP
ARABINOSYL CYTOSINE (AraC)
DNA POL INHIBITOR
CONVERTED TO TRI-PHOS FORM THEN ACTS AS CHAIN TERMINATOR
BLOCKS RIBONUCLEOTIDE REDICTASE IN CDP FORM
REPLACES RIBOSE SUGAR WITH ARABINOSE
CISPLATIN
CANCER DRUG
PLATINUM COMPLEXES CAUSE CROSSLINKING OF DNA AND TRIGGERS APOPTOSIS
AZIDODEOXYTHYMIDINE (AZT)
ANTIRETROVIRAL DRUG
NUCLEOSIDE ANALOG REVERSE TRANSCRIPTASE INHIBITOR (NRTI)
RESULTS IN CHAIN TERMINATION
SHOWS SELECTIVITY FOR HIV CELLS
EFAVIRENZ
HIGHLY ACTIVE ANTIRETROVIRAL THERAPY (HAART)
HIV
LAMIVUDINE
(3TC) HIV
REVERSE TRANS INHIBITOR
ACYCLOVIR
HERPES REPLICATION BLOCKER
SUGAR RING REPLACED BY OPEN CHAIN STRUCTURE
RESULTS IN ACYLCO-GTP HAVING HIGHER AFFINITY FOR VIRAL POL, CAUSING CHAIN TERMINATION
3 GENERAL PROPERTIES OF RNA POLS
1) REQUIRE DNA TEMPLATE
2) REQUIRE ALL 4 NUCLEOTIDE TRIPHOSPHATES AS SUBSTRATES
3) CATALYZE THE SYNTHESIS OF NEW RNA MOLECULE IN A 5' TO 3' DIRECTION
HOW DOES TRANSCRIPT INITIATION OCCUR?
REARRANGEMENT OF CHROMATIN CONTAINING THE PROMOTER SEQUENCE
BINDING OF TRANSCRIPTION FACTORS TO PROMOTER AND ENHANCER (NOT ALL GENES CONTAIN ENHANCER)
BINDING OF RNA POL TO PROMOTER FOR INITIATION OF TRNASCRIPTION
CHROMATIN REARRANGEMENT
REVERSIBLE MODIFICATIONS OF HISTONES
ACETYLATION CREATES WEAKER INTERACTION BETWEEN HISTONE(+) AND DNA (-)
LYSINE IN HISTONE --> HAT --> ACETYL-LYSINE RESIDUES IN HISTONE. HDAC REVERSES
WHAT ARE THE CHARACTERISTICS OF SPECIFIC TRANSCRIPTION FACTORS? (5)
(1) BIND DISITALLY FROM DNA PROMOTER
(2) MODULATE EFFICIENCY OF INITIATION (ENHANCER)
3) MEDIATE SIGNAL RESPONSE (e.g. HORMONE RESPONSE)
(5) CAN ACT AS CO-ACTIVATORS RESULTING IN ENHANCEMENT
WHAT ARE THE MAJOR STEPS OF tRNA SYNTHESIS?
BINDING TRANSCRIPTION FACTORS TO PROMOTER. 2 PROMOTERS WITHIN TRANSCRIBED GENE.
BINDING OF POL III
PROCESSING OF tRNA PRECURSOR (REMOVING INTRONS)
POST-TRANSCRIPTIONAL MODIFICATIONS. TRINUCLEOTIDE SEQUENCE 'CCA' ADDED TO 3' END
WHAT ARE THE MAJOR STEPS OF EUK rRNA SYNTHESIS?
BINDING OF TRANSCRIPTION FACTORS TO PROMOTERS LOCATED ON 2 DIFFERENT GENES (5.8 18 28 SEQUENCE AND 5 SEQUENCE)
TRANSCRIPTION TO MAKE rRNA PRECURSERS
REMOVING INTRONS FROM PRECURSERS
POST-TRANSCRIPTIONAL MODIFICATIONS (METHYLATION)
BINDING OF RIBOSOMAL PROTEINS TO FORM THE 2 SUBUNITS (40 & 60)
WHAT ARE THE MAJOR STEPS IN EUK mRNA SYNTHESIS?
RECOGNITION OF UPSTREAM CIS-ACTING ELEMENTS (PROMOTERS & ENHANCERS)
BINDING OF TRANS ACTING ELEMENTS (TRANSCRIPTION FACTORS)
BINDING OF RNA POLYMERASE
WHAT ARE THE GENERAL CHARACTERISTICS OF ENHANCERS (CIS-ACTING ELEMENTS)?
CANNOT INITIATE TRANSCRIPTION WITHOUT PROMOTER
FUNCITON ONLY WHEN LOCATED ON THE SAME DNA MOLECULE AS PROMOTER THEY AFFECT
CAN FUNCTION AT GREAT DISTANCE FROM THEIR PROMOTER AND IN EITHER ORIENTATION
BINDING SITES FOR ENHANCER OR SILENCER PROTEINS (TRANS-ACTING ELEMENTS OR TRANSCRIPTIONAL TRANSACTIVATORS)
WHAT ARE THE 2 TYPES OF PROMOTER SEQUENCES?
CORE PROMOTER SEQUENCES OR PROXIMAL ELEMENTS DETERMINE PRECISELY WHERE TRANSCRIPTION WILL INITIATE (TATA BOX 25 BP UPSTREAM OF START SITE)
UPSTREAM PROMOTER ELEMENTS ARE GENERALLY FIXED WITH RESPECT TO POSITION NEAR START SITE (CAAT BOX)
WHAT ARE THE EUK AND PROK TRANS-ACTING FACTORS OF mRNA SYNTHESIS?
PROKARYOTES -- CLUSTERED RELATIVELY CLOSE TO START SITE. AT-RICH SEQUENCE AT -7 TO -10 BINDS SIGMA FACTOR WHICH IS IMPORTANT FOR RNA POL BINDING
EUK -- TATA BOX BINDING PROTEIN (TBP) IS PART OF LARGE TRANS-ACTING COMPLEX CALLED TFIID.
TBP OF THE TFIID BINDS TO TATA BOX, THEN RNA POL II AND BASAL TRANSCRIPTION FACTORS (TAFs) CREATING BASAL COMPLEX.
OTHER TAFs BIND TO STABILIZE THE BASAL COMPLEX (e.g. HELIX TURN HELIX AND ZINC FINGERS)
HELIX-TURN-HELIX
REGULATORY TRANSCRIPTION FACTOR FOR EUK
2 ALPHA HELICES SEPARATED BY BETA TURN.
ONE BINDS TO SPECIFIC DNA SEQUENCE AND THE OTHER INTRACTS WITH OTHER TFs
ZINC FINGERS
REGULATORY TRANSCRIPTIONFACTOR IN EUK
ONE OR MULTI MOTIFS WITH ZINC ATOM LINKED TO 2 CYSTEINE RESIDUES AND 2 HISTIDINE SIDE CHAINS
MOTIF FLANKED BY AN ALPHA HELIX AND A BETA SHEET. HELIX BINDS TO TARGET SEQUENCE
LEUCINE ZIPPER
REGULATORY TRANSCRIPTION FACTORS IN EUK
ALPHA HELIX CONTAINING AT LEAST 4 LEUCINE RESIDUES WHICH ALIGN ON ONE EDGE
BINDS WITH ANOTHER LEUCINE HELIX VIA HYDROPHOBIC INTERACTIONS
DIMER FORMED (HOMO OR HETERO) AND BASIC SIDE HELIX ADJACENT TO ZIPPER BINDS TO DNA
HELIX-LOOP-HELIX
REGULATORY TRANSCRITION FACTOR IN EUK TRANSCRIPTION
ONE DNA BINDING HELIX AND TWO DIMERIZED HELICES SEPARATED BY A NON-HELICAL LOOP
BASIC CHARACTERISTICS OF LAC OPERON
CONSISTS OF PROMOTER, OPERATOR, REPRESSOR (SEPARATE GENE), AND 3 STRUCTURAL GENES
REPRESSOR BINDS TO OPERATOR AND BLOCKS RNA POL IN THE ABSENCE OF LACTOSE
IF LAC IS PRESENT AND GLUCOSE IS DEPLETED, LAC CONVERTED TO INDUCER (ALLOLACTOSE) WHICH INACTIVATES REPRESSOR AND STARTS SYNTHESIS OF cAMP
cAMP BINDS TO CRP, WHICH BINDS TO REGULATORY SEQUENCE
RNA POL BINDS AND TRANSCRIPTION OCCURS
GIVE AN EXPAMPLE OF TISSUE SPECIFIC REGULATION OF TRANSCRIPTION
ALBUMIN GENE IN LIVER MODULATED BY TFs
LIVER PROTEIN TRANSTHYRETIN (PREALBUMIN) ACTS AS TF SPECIFIC TO HEPATOCYTES
MEDICAL RELAVENCE OF RNA BINDING PROTEINS
NEURODEGENERATIVE DISEASES IN PRE-mRNA (POMA PARANEOPLASTIC NEUROPATHIES DUE TO MUTATION IN hnRNP-P2 WHICH BINDS TO PRE-mRNA)
WHAT ARE SOME DISEASES ASSOCIATED WITH POLY-A SIGNAL
(AAUAAA) IN mRNA?
COLORECTAL CANCER
INSULIN-LIKE GROWTH FACTOR 1 DEFICIENCY
SCID
ALPH/BETA THALASSEMIA FROM REDUCED GLOBIN CHAIN SYNTHESIS. MAJOR DEFICIENCY CAUSES
CHRONIC ANEMIA. TREAT BY BLOOD TRANSFUSION AND IRON CHELATION WITH DEFEROXAMINE
WHAT ARE SOME DISEASES ASSOCIATED WITH mRNA cleavage site?
THROMBOPHILIA (HYPERCOAGULABILITY) INCLUDING DVT AND
PULMONARY EMBOLISM PE)
DESCRIBE RNA SPLICING
snRNPs BASE PAIR WITH THE 5’ SPLICE JUNCTION AND SEQUENCE WITHIN INTRON TERMED THE BRANCH POINT (ENTIRE COMPLEX CALLED SPLICEOSOME)
5’ END IS GU AND 3’ END IS AG
2’, 5’-PHOSPHODIESTER LINKAGE BETWEEN EXON 1 AND INTRON CALLED LARIAT.
EXON 1, INTRON, AND EXON 2 JOIN AND INTRON/snRNP RELEASED
SYSTEMATIC LUPUS ERYTHEMATOSUS (SLE)
DISORDER OF SPLICING CHARACTERIZED BY DEGENERATION OF SEVERAL TISSUES AND ORGANS INCLUDING SKIN, JOINTS, KIDNEYS, AND NERVOUS SYSTEM
CONTAIN ANTIBODIES TO SELF snRNP INHIBITING RNA SPLICING (AUTOIMMUNE DISEASE)
BRCA1 GENE
DEFECT IN ALTERNATIVE SPLICING CAN CAUSE BREAST AND OVARIAN CANCER
INVOLVED IN DNA DAMAGE REPAIR
DESCRIBE CHARACTERISTICS AND ASSOCIATED DISEASES INVOLVED WITH mRNA STABILITY
ALTERING DEGRADATION OF mRNA ALSO REGULATES mRNA LEVELS
Ex, TRANSFERRIN RECEPTOR – WHEN IRON INSIDE CELL IS LOW THEN TRANSFERRIN RECEPTOR INCREASES. LACK OF IRON CAUSES IRE-BP TO BIND TO 3’UTR AND PROTECT TRANSFERRIN RNA FROM DEGRADATION
MUTATIONS IN IRON RESPONSE ATTRIBUTED TO MITOCHONDRIAL AND METABOLIC DISORDERS
TUMORS, BURKITT LYMPHOMA CAUSED BY TOO-STABLE Myc RNA WHICH IS A TRANSCRIPTION FACTOR
RNA EDITING
ADDITION OF A RESIDUES ON 3’ END OF VIRAL MRNA PROLONGS STABILITY (ex MEASLES & MUMPS)
CHANGES IN BASE-PAIR SEQUENCING CAN CAUSE MAJOR MODIFICATIONS IN ORF (ex Apo B USED TO PRODUCE 2 TISSUE-SPECIFIC PROTEINS (LIVER & INTESTINE)
DESCRIBE THE CHARACTERISTICS OF microRNA
REGULATE THE STABILITY OF MRNA
PRODUCED AS pre-miRNA THEN EXPORTED TO CYTOPLASM WHERE CLEAVED BY RNase TO MAKE SMALL 2X-STRANDED RNA
2X-RNA DISSOCIATES INTO siRNA WHICH HYBRIDIZES TO TARGET MRNA FORMING A SILENCING COMPLEX (RISC) CAUSING DEGRADATION
IF NOT HYBRIDIZED EXACTLY, TRANSLATION IS JUST INHIBITED
MAY BE INVOLVED IN ALZHEIMERS, PARKINSONS, AND SUSTAINED CARDIAC HYPERTROPHY
ANTIBIOTICS THAT INHIBIT RNA POLYMERASES
ACTINOMYCIN D & ADRIAMYCIN (DOXORUBICIN), CANCER CHEMOTHERAPY. INHIBITS BOTH DNA
REPLICATION AND RNA TRANSCRIPTION. HIGHLY TOXIC
ALPHA-AMANITIN, FROM POISONOUS MUSHROOMS INHIBITS EUK RNA POL II AND LESSER EXTENT POL III
RIFAMPICIN AND RIFAMYCIN B, INHIBIT ELONGATION BY BACTERIAL POL. TREATS INFECTION SUCH AS TB
Cancer Chemotherapeutic Agents that inhibit DNA synthesis (59)
Topoisomerase inhibitors: Camptothecin – inhibits human topoisomerase I by blocking the ligation of the DNA breaks made by the enzyme
DNA Pol Inhibitors: Arabinosyl cytosine (AraC, cytosine arabinoside) Chain terminator: lacks a reactive 3’ OH so chain elongation stops when DNA Pol incorporates it into the replicating strand
Agents that cross link strands: cisplatin
Antibacterial agents that block DNA synthesis (2)
§
Novobiocin: inhibits bacterial topoisomerase (selective for bacteria)
§
Nalidixic acid: inhibits bacterial topoisomerase, treats MRSA and anthrax
Antiviral drugs that block DNA synthesis (59)
Reverse Transcriptase inhibitors:
Azidodeoxythymidine (AZT) and 2’,’3-dideoxycytidine (DDC)
Lack reactive OH’s so they stop chain elongation
Combined with other drugs for HAART (highly active retroviral therapy)
Have preferential affinity for RT and less for the host cell’s DNA polymerase
Efavirenz – non-nucleotide RT inhibitor (NNRTI) – binds allosteric site to distort the catalytic asp residues and make RT nonfunctional
Acyclovir – used to block Herpes virus (HSV)
PO’d to monophosphate form by VIRAL thymidine kinase, then to di- and tri-
PO’d nucleotide is incorporated and acts as chain terminator
Specific only for the VIRAL kinase….very little side effects if HSV is not present