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71 Cards in this Set
- Front
- Back
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The difference between bases, nucleosides, and nucleotides |
Base: A,T,C,G (know their structures) Nucleoside: sugar+base(bonded to 1' of sugar) Nucleotide: nucleoside+phosphate(bound to 4') |
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Factors stabilizing the DNA helix |
sequence-dependent base stacking pH (physiological=7.4) salt concentration temperature |
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Major and minor grooves |
Twisting of helix forms grooves along the surface large groove= major; small=minor important for things interacting with DNA |
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Intercalating agent |
an agent that can insert between the planar bases of DNA example: doxorubicin |
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Directionality of DNA |
5' to 3' dictated by sugar the two strands are complementary and antiparallel |
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Chemical properties of DNA |
nucleic acids absorb light at 260nm can quantify nucleic acids by how much light they absorb [DNA]= A260 X 0.05mg/mL/A260 |
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DNA denaturation and renaturation |
Denaturation=DNA can come apart - first comes apart at T and A rich areas - can be denatured with heat (PCR) Renaturation/annealing= hybridization to complementary bases - can tag oligonucleotides to use as probes |
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Chemical components and structural features of RNA |
Linear polymer of ribonucleotide phosphates Chemically similar to DNA Much less stable than DNA (because of 2' OH) Has uracil (instead of T) Has modified nucleotides 2'OH on ribose sugar Molar ratio of (A+U) and (G+C) not equal Some have autocatalytic activity-> ribozymes |
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Differences between DNA and RNA |
No 2'OH on DNA sugar Molar ratio of (A+U) and (G+C) is equal in DNA RNA is single stranded DNA is more stable No modified nucleotides in DNA DNA has T, RNA has U |
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mRNA unique characteristics |
Direct carrier for genetic information from DNA for translation Has 5' cap and introns Mature through post-transcriptional modifications Has a short half-life -> regulation Can be splice variants: multiple mRNA produced from a single gene Serves as a template for cDNA (retroviruses) |
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rRNA unique characteristics |
80% of all RNA Metabolically stable Part of protein synthesis apparatus-> combines with proteins to form a ribosome Processed in the nucleolus not nucleus 80S rRNA in eukaryotes; 70S in prokaryotes Has modified bases |
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tRNA unique characteristics |
Two key roles: - activating amino acids (each tRNA transfers only 1 amino acid) -recognizing codons in cellular mRNA Intermediary between DNA and proteins Have many modified bases -modified post-transcriptionally -ex. thymine put into RNA |
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mtRNA (mitochondrial RNA) characteristics |
Made by mitochondrial-specific RNA polymerase Mitochondria contain DNA and can synthesize their own proteins Code for proteins with specific function inside mitochondria |
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lncRNA (long non-coding RNA) |
About 80% of transcripts are non protein coding Are diverse and have multiple functions Serve as precursor to micro RNA |
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miRNA (micro RNA) |
Inhibit gene expression by inhibiting mRNA translation or by promoting mRNA degradation Are the basis for some nucleic acid based forms of therapeutics and are targets of therapeutic agents |
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Retroviruses |
Have RNA genome: Infect a cell, then release the RNA genome Use reverse transcriptase to make cDNA which gets integrated into the nucleus-> have provirus -this requires integrase Viral genes are transcribed and translated to form viral proteins and new viral particles |
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What is DNA replication? |
The duplication of DNA necessary for two cells to divide with the same genetic information |
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Components of the DNA replication fork |
(both parental strands copied at the same time) Helicase: unwinds helix ahead of fork SSPBs: prevent strands from reassociating Topoisomerase: break phosphodiester bonds to relieve super-coiling Primase: synthesize primers -also have enzymes that remove primers |
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How a primer functions (DNA replication) |
Primer= has 3' OH off of which a polymerase can bind (DNA pol requires a 3'OH to function) - is an RNA oligonucleotide -synthesized 5' to 3' by Primase DNA pol adds on to 3'OH and keeps adding |
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Components of replication on the leading and lagging strands |
Polymerases: add nucleotides to strand growing 5' to 3', copying DNA template - different for Prok.'s and Euk.'s Ligase: joins 2 adjacent DNA strands Enzymes to remove primers |
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Prokaryotic DNA Polymerases |
Polymerase I: fills gap after RNA primer removal; also DNA repair Polymerase II: DNA repair-> 3' to 5' exonuclease activity Polymerase III: Replication, synthesis of DNA 5' to 3'; also 3' to 5' exonuclease |
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Eukaryotic DNA Polymerases |
Polymerase Epsilon: synthesis of leading strand; repair Polymerase Delta: synthesis of lagging strand (Okazaki fragments); also repair and gap filling after primer removal Polymerase Alpha: primase; primes for Okazaki fragments |
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Enzymes that remove primers |
RNase H: hydrolyzes RNA of DNA-RNA hybrids Flap Endonucleases 1 (FEN1): recognize unannealed portion of RNA near 5' end of primer and cleave downstream in DNA region of primer |
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Telomere |
end of the chromosome consist of repeating sequence of bases |
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Telomerase |
Acts as RNA-dependent DNA polymerase Contains RNA with complementary copy of repeating sequence in telomere - pairs with 3' overhang -uses its own RNA as a template to make DNA to lengthen the 3' end of the DNA |
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SNP |
Single Nucleotide Polymorphism genomic variations that all people are born with -inherited One nucleotide difference |
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Mutation |
difference in DNA from when the person was born something happens to the DNA - damage |
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Types of DNA damage (examples of damage) |
oxidative deamination spontaneous depurination methylation thymine dimers created |
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Types of mutations |
Missense (technically a variation)= amino acid switched Nonsense= amino acid in coding region is now a stop Insertion= causes frameshift; changes amino acids or causes early stop Deletion= causes frameshift also |
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Sources of DNA mutations |
Random errors in replication Errors during DNA damage repair Alterations by cellular constituents (reactive oxygen species) Environmental changes (radiation, UV light, ionizing radiation) |
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Base excision repair |
DNA glycolases Recognize small distortions in DNA from damage to a single base Glycolase cleaves bond of base and other enzymes restore to normal |
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Nucleotide excision repair |
endonucleases recognize bulky distorted regions cleave large area and remove distorted region polymerase fills gap, adding to 3' end of cleaved DNA -join with ligase |
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Mismatch DNA repair |
mismatched=bases that don't form normal Watson-Crick base pairs mismatch is recognized by enzymes -parental strand is methylated incorrect base put in during replication |
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MGMT mediated repair |
Methyl Guanine Methyl Transferase Direct demethylation when guanine is methylated, C won't bind but T will reversed by MGMT |
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Photoreactivation |
UV-induced cyclobutane pyrimidine dimers repaired by photolyase and light primarily in bacteria |
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Bypass synthesis |
Replication coupled repair DNA delta polymerase gets stuck at a lesion nucleotides are incorporated opposite the lesion by polymerases with relaxed stringency that bypass the lesion |
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Daughter strand gap repair |
Replication Coupled Repair a gap is left in new DNA at the point of damage part of the parental strand goes through recombination to fill in gap in daughter new gap in parental strand is filled in by DNA polymerase |
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Homologous recombination |
genetic exchange between 2 molecules that share an extended region of almost identical sequence can happen in females between X chromosomes important in pharmacogenomics |
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Transposable elements |
Pieces of DNA in genome want to move to new area Studied by Barbara McClintock "Jumping genes" |
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DNA as a template for RNA transcription |
DNA template copied 3' to 5' mRNA synthesized 5' to 3' |
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Post-transcriptional processing of mRNA |
first transcribed-> hnRNA (hetergenous nuclear RNA) Rapidly acquires poly(A) tail Intron splicing 5' Cap |
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mRNA: Capping |
5' Cap added during post-transcriptional
At 5' end, initial nucleotide is a pyrimidine with 3 phosphates -One phosphate comes off GTP binds, keeping 1 of its phosphates and is methylated -Methylguanosine Ribose 2' hydroxyl is also methylated |
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mRNA: Addition of poly(A) tail |
RNA pol transcribes stop codon and continues past to polyadenylation signal Enzymes cleave the hnRNA forming 3' end Poly(A)tail is added to the 3' end by poly(A) polymerase Poly(A) tail is a protein binding site that protects from degradation |
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Definition of gene |
a locus of DNA that encodes a functional RNA or protein product it is a unit of heredity that is passed form parent to offspring |
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Genomic differences between Eukaryotes and Prokaryotes |
Eukaryotes: 23 chromosome per haploid, linear DNA, have histones, diploid somatic cells, haploid germ cells, 3e9 base pairs per haploid cell, 64% unique and 35% repetitive genes, no operons, mRNA is not polycistronic and has introns Prokaryotes: 1 chromosome per haploid, circular DNA, no histones, no diploids, all are haploid, 4e6 base pairs per cell, 100% unique genes, have operons, mRNA is polycistronic with no introns |
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Gene structure in Eukaryotes |
Promoter- 5' UTR- initial exon with start codon in it- alternating series of introns and exons- terminating exon with stop codon- 3' UTR- Poly(A) signal |
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Eukaryotic RNA Polymerases |
RNA polymerase I: Ribosomal RNA's in nucleolus RNA polymerase II: messenger RNA's in nucleus; helped by Basal transcription complex RNA polymerase III: 5S ribosomal RNA, small cellular RNA's, and viral RNA's (prokaryotes only have one RNA pol) |
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Transcriptional initiation signaled in the promoter through a protein complex |
Protein complex= Basal transcription complex (pre-initiation complex) TATA binding protein (TBP) is part of TFIID-> TBP binds first, then TFIIA and TFIIB interact creating a complex RNA Pol II binds the complex, TFIIE, TFIIF, TFIIH bind, cleaving ATP and initiating gene transcription There are also co-activators in TFIID that can bind other regulatory proteins, increasing rate |
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Chromatin structure |
Naked DNA: all histones removed; how it exists in cells Nucleosome: DNA wrapped around a histone; not transcriptionally active once compacted; histone octamer Solenoid: helical winding of at least 5 nucleosome strands, not transcriptionally active |
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Topoisomerase I |
works by making a transient cut in one DNA strand passes unbroken strand through break rejoin the broken ends |
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Topoisomerase II |
works by breaking both DNA strands passes a DNA segment through the break rejoin the strands |
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Preinitiation complex in eukaryotes |
TFIID/TBP binds-> DNA bending occurs-> other TFII proteins are recruited to the initiation complex to form a complex for RNA pol II to bind and initiate transcription RNA polymerase needs help |
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Basic Principles of transcription factors |
Alter gene expression Many different ones bind to DNA Bind to specific DNA sequence called the response element, which is a cis- element Can be large proteins- part interacts with other factors and part interacts with DNA They are trans-acting and are proteins very complex and important |
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Role and Function of Punctuation codons |
(stop and start) AUG is the start codon and codes for methionine Three stop codons: UAG, UAA, UGA - don't code for an amino acid -termination or nonsense codonse |
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Aminoacetylation |
Amino acids must be linked to their appropriate tRNA carriers before being incorporated into proteins Catalyzed by aminoacyl-tRNA synthetases - each is specific for a single amino acid and tRNA A multi step reaction that requires ATP |
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Specificity and Fidelity of aminoacetylated tRNA's + tRNA structure |
Each synthetase must correctly recognize one to several tRNA species that carry the same amino acid Structural elements: -anticodon: base pairs via H bonds -elements of acceptor stem -parts of variable loop or D-stem loop |
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Nomenclature of tRNA |
acceptor specificity: superscript Activated/charged: amino acid before tRNA ex: Met-tRNA(met) |
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Wobble pairing |
a normal anticodon can pair with up to three different codons for the same amino acid Wobble can occur in pairing of the first base (5') of the anitcodon with the 3rd base (3') of the codon For utility and efficiency |
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Prokaryotic ribosomal subunits |
Small= 30S Large= 50S Monomer size= 70S |
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Eukaryotic ribosomal subunits |
Small= 40S Large= 60S Monomer= 80S |
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eIF2 |
(eukaryotic initiation factor) Regulator of the initiation step in protein synthesis When it is phosphorylated, it is inactive and protein synthesis cannot begin Met-tRNA(met) forms a complex with eIF2, which binds GTP and binds the small ribosomal subunit |
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eEF-1Alpha |
(eukaryotic elongation factor) Ensures codon- anticodon binding is correct GTP-binding alpha subunit -activated for association with other proteins when it has GTP Incoming aminoacyl-tRNA first combines with eEF-1Alpha before binding the the mRNA-ribosome complex |
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eEF2 |
(eukaryotic elongation factor) involved in translocation Complexes with GTP and binds to ribosome, causing a conformational change that moves mRNA and base-paired tRNA |
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eIEF4 |
Triage-> prioritizes messages mRNA isn't just released from nucleus to find ribosome on its own Some proteins need to be prioritized and made ASAP Associates with many factors in the cell then interacts with ribosome Highly regulated by stress or drugs - crisis mode down-regulates |
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How prokaryotic protein synthesis is different than eukaryotic |
mRNA can be polycistronic Specific initiation tRNA is formylated methionine: fmet-tRNA(met) Ribosome is less complex No nucleus-> no orientation when coming out of nucleus mRNA oriented by Shine-Dalgarno sequence upstream of start codon and a sequence in the 16S rRNA binding |
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A site |
Acceptor site: accepts new transfer RNA Aminoacyl site Starts with second aminoacyl-tRNA binding and base pairing with 2nd codon (first binds to P site) |
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P site |
Translocation Peptidyl site Binds first aminoacylated tRNA (Met-tRNA(met)) Peptidyl-tRNA translocates to P site |
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E site |
Exit site In prokaryotes, binds t-RNA after it is displaced from P site |
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Initiation |
Complex between the small 40S subunit, an mRNA, a tRNA, followed by association of the 60S subunit to form an 80S ribosome |
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Elongation |
Stepwise formation of peptide bonds |
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Termination |
Completed polypeptide is released from its tRNA and the ribosome |
