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23 Cards in this Set
- Front
- Back
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Transcription and RNA - overview! |
A gene is a segment of DNA that codes for a protein (via mRNA) or another functional RNA. |
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How does the prokaryotic RNA polymeraseknow where to start?! |
in e. coli one of several sigma factors associates with the single rna polymerase to increase the affinity of RNAP for these promoter sequences that are just upstream of the initiation site |
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Transcription |
transcription produces RNA in the 5'->3' directionNO PRIMER but chemically same thing as REPLICATION-3'->5'template strand=antisense=noncoding=negative (-) bottom-(upstream) 5'->3' (downstream) nontemplate=sense=coding=positive(+) -prokaryotes such as E. coli have a single RNA polymerase -humans have three RNAP's RNAP1: produces ribosomal rRNA RNAP2: produces messenger mRNARNAP3: produces rRNA and transfer tRNA eukaryotic RNA is transcribed with EXONS that code for the polypeptide sequence and introns (NONCODING=removed during/after transcription) |
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4611 Chapter 21 slides powerpoint.ppt How does the eukaryotic RNA polymeraseknow where to start?! |
4611 Chapter 21 slides powerpoint.ppt Eukaryotes have a variety of upstream and downstreamelements that may vary from gene to gene. Generaltranscription factors bind some combination of theseelements and recruit RNAP to the start site.tata box is further upstream than prokaryotes (31-35) |
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What distal regulatory elements affect transcription? |
4611 Chapter 21 slides powerpoint.ppt Enhancers and silencers are DNA sequences that helpregulate transcription. |
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Enhancers |
Activators are going to promote RNAP2 localization to the start site activators are proteins that often interact with the enhancer sequence |
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Silencers |
-Repressors are proteins that decrease RNAP activity -Activators bind coactivator called mediator to link transcription factor signal to transcription |
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4611 Chapter 21 slides powerpoint.ppt What general transcription factors arerequired to initiate transcription?! |
4611 Chapter 21 slides powerpoint.ppt Eukaryotic transcriptioninitiation requires 5 highlyconserved generaltranscription factors (TFIIB,TFIID, TFIIE, TFIIF, andTFIIH). General TF help RNAP2 locate the transcription start site and open up the transcription bubble |
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4611 Chapter 21 slides powerpoint.ppt How is the RNA transcript elongated? |
-Phosphorylation of the c terminal domain of RNAP and binding at elongation factors changes the conformation of RNAP so it can leave the start site and make a full length transcript |
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4611 Chapter 21 slides powerpoint.ppt How does RNA Polymerase transcribe DNA? |
the dna template strand enters RNA polymerase and forced to make a 90 degree turn to exit this turn forces a template base into the active site where it is read and basepaired to some nucleotidetriphosphate that enters through the funnel |
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4611 Chapter 21 slides powerpoint.ppt w does RNAP proofread transcripts? |
Newly synthesized strand of RNA forms an RNA-DNA hybrid HELIX with the DNA TEMPLATE strand within your RNAP if the incorrect nucleotide is incorporated the helix gets DISTORTED and RNAP backtracks pushing the recently adding bases out of the funnel where they are removed by RNAP proofreading activitingRNAP has a rate of 1/10000 Bp |
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4611 Chapter 21 slides powerpoint.ppt How is transcription terminated in E. coli?! |
In e.coli transcription may be intrinsically terminated when RNAP transcribes a GC region forming a HAIRPIN to pull a subsequent U-Rich region off of the DNA template with rho dependent termination the rho protein factor specifically binds to a C-rich sequence on the transcript and moves toward your RNA polymerase eventually bumping it off of the transcript of the DNA template |
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4611 Chapter 21 slides powerpoint.ppt How can organisms regulate the expressionof some groups of related genes?! Bacteria: operons consist of several genes under thecontrol of one promoter that are transcribed as one mRNA. Eukaryotes: related genes may be grouped together underthe control of the same regulatory elements, but aretranscribed separately.Smallest Kd = binds tightest = best inhibitor |
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4611 Chapter 21 slides powerpoint.ppt ow is the 5ʼ end of eukaryotic mRNAsprotected from exonucleases?! |
4611 Chapter 21 slides powerpoint.ppt ukaryotic mRNAs are capped at the 5’end by linking aguanosine residue to the emerging mRNA via a 5’-5’triphosphate linkage. !no free 5' end for exonucleases to attack |
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4611 Chapter 21 slides powerpoint.ppt How is the 3ʼ end of eukaryotic mRNAsprotected from exonucleases?! |
4611 Chapter 21 slides powerpoint.ppt Eukaryotic mRNAs arepolyadenylated at the 3’ end. poly(A) binding proteins protect themRNA from degradation and helpcontrol the mRNA lifetime.! recruits additional proteins for protectionshort polyAtail=short lifespan of mRNA |
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Wh 4611 Chapter 21 slides powerpoint.ppt at happens to introns?! |
4611 Chapter 21 slides powerpoint.ppt ntrons are removed and the exons are spliced together toform the mature mRNA product.! introns are noncoding, spliced out cannot be encoded in the transcription process |
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wh 4611 Chapter 21 slides powerpoint.ppt at happens to introns? |
loops=introns |
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4611 Chapter 21 slides powerpoint.ppt How does the cell know where to splice theexons?! |
4611 Chapter 21 slides powerpoint.ppt Splicing occurs at conserved sequences at the exon-intronjunction.! GUAG |
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4611 Chapter 21 slides powerpoint.ppt w does the cell remove introns? |
4611 Chapter 21 slides powerpoint.ppt Splicing occursthrough twotransesterificationreactions catalyzedby an RNA-proteincomplex called thespliceosome orcatalyzed by theRNA itself. |
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TRANSESTERIFICATION RXN |
additional hydroxyl does a nuc attack breaking phosphodiester bond then in step two there is a 3' OH to attack exon 2 |
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4611 Chapter 21 slides powerpoint.ppt can RNA act as an enzyme?! RNA has multiple functional groups and adopts unique 3-dimensional conformations just like a protein enzyme.! |
Ribozymes Guanosine free OH nuc attacks |
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4611 Chapter 21 slides powerpoint.ppt at is the advantage of splicing?! |
4611 Chapter 21 slides powerpoint.ppt Most human structural genes undergo alternative splicingwhere different processing of the transcript produces variantmature mRNAs.! Introns can be regulatory moleculesIntrons may be advantageous for modification, fewer genes needed--> splicing leads to more variationdifferent protein products from same gene due to different splicing in different tissues |
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4611 Chapter 21 slides powerpoint.ppt at is RNA interference (RNAi)?! Cells have amechanism forrecognizing dsRNAand using it todestroy relatedmRNAs.! |
mRNA and RNA can be separated by affinity column using poly T O.S. that would bind to Poly A tails of mRNA-RNA interference is useful for REGULATION of gene expression as well as for VIRAL DEFENSE -exploited in the lab, manipulated to investigate gene function and potentially generate therapeutics -dicer enzyme (chop up RNA) recognizes exogenous (outside cell) RNA duplexes and clloses them into 22 BP double stranded RNA fragments called short interacting…siRNAcell also produces microRNA miRNA that fold themselves into 22 BP HAIRPINS into siRNAs-22 bp duplexes of siRNA/miRNA are recognized by RNA induced silencing complex (RISC) RISC separates the strands (Guide strand remains bound to RISC while the passenger strand is degraded RISC then uses guide strand to locate, bind, and degrade any complementary mRNA to limit gene expression |
