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27 Cards in this Set
- Front
- Back
Features of EUK mRNA
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5' cap (m'GTP), 5'UTR, AUG start codon, ORF, STOP codon (UGA, UAG, UAA), 3'UTR, polyA tail
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importance of KOZAK sequence in EUK
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optimal, most efficient start codon sequence: ACCAUGG. need to know sequence in order to design primer to amplify product in lab.
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translational frameshifting
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the act of shifting the reading frame. could be due to fusion protein, and sometimes can be due to the secondary structure of mRNA. It has been described mainly in viruses (especially retroviruses), retrotransposons and bacterial insertion elements, and also in some cellular genes.
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hydrolytic editing of tRNA
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accuracy measure performed by trna. editing site double checks that the correct aa is charged on tRNA. it must fit editing site or will be removed before being added to a.a. chain by tRNA synthetase
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nonsense mediated mRNA decay occurs when?
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When exons and introns do not splice properly, causing introns to remain in mrna, and exon proteins to be missing. This is recognized when the mRNA is released in the cytosol, step loop structure forms and mRNA is degraded
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Gel Mobility Shift Assay
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Protein binds to DNA, which slows movement over electrophoretic gel. Can analyze shift to determine protein binding
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CHiP assay (immunoprecipitation)
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use formaldehyde to sheer dna from histone. Add antigen to purify protein, then use PCR to amplify isolated DNA sequence.
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DNA affinity chromatography
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add total cell proteins to column with cDNA on beads. Then wash with low salt conc. to remove proteins that did not bind. Medium salt wash to isolate proteins that bind. REpeat in new column, with specific cDNA sequence of interest. Proteins that bind to sequence will be captured, and then follow with med salt wash to remove prots that didn't bind. Then protein of interest can be isolated with High Salt wash
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DNAse I footprinting
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Label DNA with P (to help determine sequence with Gel Electrophoresis). Add Specific binding protein, and then follow with DNAse. Run on gel electrophoresis, there will be DNA missing from where binding protein was located=Can determine sequence that Protein binds to.
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Reporter assay can be used to measure _____activity. ________ site will not show transcription if proposed recognition site does show transcription.
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Transcriptional.
Mutated. |
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Characteristics of RNA polymerases
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Reads 3'-5' to synthesize 5'-3'. Either strand of DNA dble helix can serve as a template for RNA synthesis. TFs needed to initiate transcription. RNA polymerases do not possess proofreading activity/no nuclease activity.
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Structure of EUK Ribosome
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28S rRNA + 5.8S rRNA + 5S rRNA= 60S subunit (top)
18S rRNA + 35 proteins= 40S subunit (bottom). Complete Ribosome is called an 80S |
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RNA pol I transcribes multiple copies of_________ and identical sets of the rRNA genes.
______ promoters allow multiple RNA pol I to load and transcribe, found in _______ spacers. Separation between functional products are referred to as __________. |
Tandemly repeated.
Multiple. Nontranscribed spacers. Transcribed spacers. |
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rRNA post transcriptional processing.
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cleavage (not splicing), and methylation. Then mature rRNA associates with proteins and becomes subunits. 5S MADE BY POL III!
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RNA pol II characteristics
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CTD tail (imp for modifications), promoter sequence, TFs, initiation complex needed to recruit and stabilize pol II.
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Enhancer elements
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can be far away from promoters to stimulate transcription of a gene/genes. Enhancers may be on the 5' side of 3' side of a gene, occasionally in an intron. Enhancer elements function independent of orientation with respect to a gene, but are "cis acting". Enhancer elements are binding motifs for activators (TFs).
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Promoters
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are "cis acting". TFs bind to either promoters or enhancers. TATA boxes, CG boxes are common promoters. Help position pol II on +1 of transcriptional unit.
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Housekeeping genes have _______ promoters, while most genes restricted to differentiated cell have _________.
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CG boxes.
TATA boxes. |
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C terminal Domain of RNA pol II
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52 tandem repeats of 7 a.a. Serine 5 is phosphorylated by TF IIH, which loads RNA processing components to pol. Ser 2 is extensively phosphorylated which facilitates splicing factor binding. TFs bind to sequences and other TFs to position pol II to the gene to initiate transcription.
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Role of CTD in mRNA processing
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pSer5= 5' capping
pSer2 + pSer5= Splicing pSer 2= Poly A tail processing |
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Positive versus Negative regulation
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Positive regulation is when repressor binding turns gene on. Repressor is active or inactive by addition of ligand.
Negative regulation is when bound repressor shuts gene off. Repressor can be active or inactive, by addition of ligand. |
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Positive versus Negative Feedback loop
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Positive feedback increases transcription of gene in response to stimulus. Negative feedback degreases transcription in response to stimulus.
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Feed forward loop
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ex: A activates B, A and B activate C
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Steps of transcription initiation in RNA pol
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insert here
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steps of translation initiation in Ribozome
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insert here
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steps of translation termination
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insert here
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levels of control exhibited by transcription and translation:
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transcriptional control, RNA processing control, RNA transport and localization control, mRNA degradation control, translation control, protein activity control
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