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69 Cards in this Set
- Front
- Back
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Who proposed the central dogma of molecular biology?
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Francis Crick
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What was the central dogma as proposed by Crick missing?
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RNA processing
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What are RNA polymerases?
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Enzymes that catalyze the copying of DNA into an RNA transcript
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What does RNA polymerase I do?
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Transcribes ribosomal RNAs
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What does RNA polymerase II do?
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Transcribes all mRNA's and some small RNA molecues (small nuclear RNAs or snRNAs) that play a role in RNA processing
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What does RNA polymerase III do?
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Transcribes transfer RNAs (tRNAs) and small ribosomal RNAs (rRNA)
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What are the 4 types of RNAs?
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Ribosomal, small stable, messenger and heterogeneous nuclear, and small non-coding
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What percentage of total RNA does rRNA represent?
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75% (most abundant RNA)
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What are the types of small stable RNAs?
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Transfer RNA (tRNA), small nuclear RNAs (snRNAs- involved in splicing), and 5S ribosomal RNA (5S rRNA)
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What percentage of total RNA do small stable RNAs represent?
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15%
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What percentage of total RNA do messenger RNAs and heterogeneous nuclear RNAs represent?
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10%
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What do small non-coding RNAs do?
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They have regulatory roles
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What are regulatory sequences in DNA?
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Specific sequences of DNA that bind specific proteins that regulate transcription; not transcribed into RNA
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What is the promoter?
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Regulatory region upstream from the gene and relatively close to the region on the gene where transcription starts
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What are enhancers?
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Regulatory DNA sequences that modulate transcription from a distance
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How do promoter and enhancer regions regulate transcription?
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They bind specific DNA regulatory (or binding) proteins
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How is gene expression controlled in some single celled organisms?
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Temperature and nutrient levels
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How is gene expression controlled in multicellular organisms?
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1. Direct cell to cell contact
2. Proteins that are secreted from cells into the blood or interstitial fluids such as growth factors or hormones |
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What are the levels of control of gene expression?
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-Transcription (control over mRNA production)
-RNA processing control -RNA transport control -Translation (protein synthesis control) -Protein turnover (protein inactivation and protein degradation) |
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What are RNA polymerases?
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Enzymes that perform transcription
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How does RNA polymerase work?
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It moves stepwise along the DNA, unwinding the helix to expose a new region of template strand for complimentary base pairing; substrates are ribonucleoside triphosphates- ATP, GTP, CTP, UTP
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How does the immediate release of a newly transcribed RNA affect RNA transcription?
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Many RNA copies can be made from the same gene quickly, with the new RNA molecules beginning before initial ones are completed
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How fast can RNA polymerase synthesize transcripts?
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20 nucleotides/sec
>1000 transcripts can be made in 1 hour from a single gene |
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How do bacteria recognize where on the genome to start transcribing?
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1 RNA does it all- mRNA, structural and catalytic; sigma factor helps holoenzyme recognize promoter
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How does eukaryotic RNA polymerase II relate structurally to bacterial RNA polymerase?
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It is larger and has 12 subunits instead of 5 (in bacteria)
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How are RNA polymerase II and bacterial RNA polymerase similar?
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Structurally similar and share some common subunits
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What are the two major differences between RNA polymerase II and bacterial RNA polymerase?
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1. Bacterial RNA polymerase can begin transcription on a DNA template in vitro w/o the help of other proteins. RNA polymerase II requires the help of a set of proteins called transcription factors to begin transcription
2. Transcription in eukaryotes has to deal with DNA packing into both nucleosomes and higher levels of DNA packing which do not occur in bacterial chromosomes |
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What are general transcription factors?
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They assemble on ALL promoters used by RNA polymerase II
-help position the polymerase correctly on the promoter -separate the strands of DNA -release the polymerase from the promoter into elongation mode -designated TFII, TFIIA, TFIIB etc |
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How does transcription initiation in a eukaryotic gene by RNA polymerase II begin?
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TFIID binds to a DNA sequence in the promoter called the TATA box
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What is the TATA box?
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Short double stranded DNA sequence made of mostly of thymine and adenine nucleotides, approx 25 nucleotides upstream from the state site of transcription
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Which DNA sequence that signals the start of transcription is most important?
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TATA box
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What do RNA pol II promoters that don't have a TATA box contain?
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Strong INR
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What is the TBP?
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TATA binding protein, subunit of TFIID recognizes and binds the TATA box
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What happens after binding of TFIID to the TATA box?
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Adjacent binding of TFIIA and TFIIB at the promoter
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What happens after the binding of TFIIA and TFIIB?
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Transcription factors TFIIE, TFIIF, TFIIH and other assemble with RNA pol II at the promoter
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What is the Transcription Initiation Complex?
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Transcription factors and RNA pol II
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What does transcription factor TFIIH contain?
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A DNA helicase and a protein kinase
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What does TFIIH do?
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Uses the energy of ATP hydrolysis to unwind and open the DNA double helix; uses its kinase to phosphorylate RNA pol II
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What does the phosphorylation of RNA pol II cause?
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A change in conformation, releases it from the other general transcription factors so it can begin elongation
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What are transcriptional activators?
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Proteins that (not TFs) bind to enhancer sequences to attract RNA pol II to the transcription start site; typical genes have many
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What do transcriptional activators do?
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Help RNA pol II and TFs bind to DNA that is packaged into chromatin, can act from distances of many thousand nucleotide pairs away
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What is a mediator?
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A multiprotein complex required for transcription; allows activator proteins to communicate correctly with RNA pol II and the TFs
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What are chromatin remodeling enzmyes and histone acetylases?
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Enzymes required for eukaryotic transcription that allow transcriptional machinery greater access to DNA and facilitate the transcription process
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How does RNA pol II proceed through elongation?
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In a jerky pattern, pauses at some sequences and proceeds rapidly through others
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What are elongation factors?
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Special proteins that keep the polymerase attached to the gene until transcription is complete
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What do elongation factors do?
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Associate with the polymerase shortly after initiation and help the polymerase move through different types of sequences
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What is super coiling?
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RNA pol II proceeds along the DNA strand of a double helix it creates superhelical tension
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What is topisomerase?
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Enzyme that relieves superhelical tension
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What happens when RNA pol II has made ~25 nucleotides of RNA?
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5' end is modified by addition of a methyl guanosine cap (RNA capping)
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What enzymes perform RNA capping?
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1. Phosphatase removes a phosphate from the 5' end of RNA
2. Guanyl transferase adds a GMP 5' to 5' 3. A methyl transferase adds a methyl group to guanosine |
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What is the CTD?
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Carboxyl terminal domain
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What is the composition of CTD of RNA pol II?
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Composed of a repeated amino acid sequence- 7aa's long repeated 52 times with 2 serine residues in each repeated sequence
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What is the mechanism of CTD of RNA pol II?
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-Phosphorylation by TFIIH on serine 5 residue results in binding of capping proteins to CTD, capping occurs as soon as 5' end emerges
-Next the tail is extensively phosphorylated on Ser 2 positions which attract splicing and 3' end processing proteins and positions them to act on the RNA as it lengthens |
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What is RNA splicing?
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Removal of introns from precursor mRNA
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What are the enzymes that catalyze the splicing reaction?
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5 additional RNA molecules, more than 200 proteins, numerous ATP molecules
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What is the RNA splicing reaction?
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1. Adenine nucleotide in intron sequence attacks the 5' splice site with 2' OH group, cutting RNA backbone
2. THe cut 5' end of intron gets bonded to 2' OH of adenine nucleotide (5'-2') creating a loop 3. The free 3' OH of exon reacts with start of next exon 4. This joins the exon and releases the intron as a lariat |
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What 3 positions on an RNA molecule are required for intro sequence removal?
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-5' splice site
-3' splice site -Branch point in intron that forms lariat |
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What tells where splicing to occur?
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Consensus nucleotide sequences
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What is the consensus nucleotide sequence?
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Signal beginning and end of an intron
Only 3 blocks of nucleotide sequences are necessary to remove an intron Only GU at start and AG at end of intron sequence are invariant The remaining positions can be occupied by various other nucleotides |
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What is alternative splicing?
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Splicing of a transcript of a particular gene to produce different sets of mRNAs and therefore different proteins
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What performs RNA splicing?
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Spliceosome
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What is the spliceosome composed of?
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RNA molecues- recognize intron-exon beginning and end points, 5 RNAs that perform this function ~<200 nucleotides
each named U1, U2, U4, U5, U6; snRNPs |
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What is an snRNP?
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Small nuclear riboprotein, form spliceosome
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How does the spliceosome work?
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Breaks and replaces base-pair interactions check and recheck RNA sequences before reactions are allowed to proceed
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What does breaking and replacing base-pair interactions by the spliceosome accomplish?
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1. Identifies consensus sequence
2. Brings 2 ends of intron together 3. Provides enzymatic activity for 2 rxn steps |
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Which consensus sequences are deposited on the 3' end of the RNA molecule as it leaves RNA pol II
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The hexamer AAUAAA, GU rich element, CA sequence
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Which consensus sequence is bound by CstF?
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A GU rich element
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What is the consensus sequence bound by CPSF (cleavage and polyadenation specificity factor)?
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The hexamer AAUAAA
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What are the steps of polyadenation of RNA?
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1. CstF + CPSF bind to consensus sequences
2. Additional cleavage factors are added and RNA molecule is cleaved 3. RNA pol II continues to transcribe and eventually dissociates from the DNA template 4. Poly-A polymerase adds ~200 adenine nucleotides to the cleaved 3' end (no template required) 5. Poly-A binding proteins assemble and determine lenth of tail- remain with mRNA and assist with translation |
