B M B 251: EXAM 3
72 Cards in this Set
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What is required for transcription initiation in eukaryotes?
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-assembly of RNA polymerase II and binding of general transcription factors (GTFS) on the core promoter
-note that GTFS are analogous to the sigma factors in bacteria
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14. What is required to open the transcription bubble in prokaryotes?
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Prokaryotes: One RNA polymerase. Bacterial RNA pol only requires sigma factor to recognize and bind to the promoter
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14. Eukaryotic RNA pol II requires at least 5 additional proteins to associate with a promoter. Which protein recognizes the promoter sequence? Which protein is required to open the transcription bubble? What is/are the analogous protein(s) in prokaryotes?
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TFIID recognizes the promoter sequence (the starting point of transcription) TFIIH is required to open the transcription bubble (similar to a helicase and a kinase) TATA Box (TBP protein->GTF) is analogous to the sigma factor binding site in prokaryotes In a broad sense, eukaryotic GTFs c…
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14. What is a TATA box?
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Located upstream of transcription start site It phosphorylates Ser5 (serine in 5th position) of RNA polymerase CTD Releases RNA polymerase from the promoter Phosphorylation is used as a signal for transcription
a short DNA sequence within the promoter region. TATA box allows RNA polymera…
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14. Relative to one another, what are the locations of a gene's: promoter (multiple elements), exons, introns, terminator?
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Promoter: 5' end of gene (start of initiation) upstream of the gene Introns: Middle (lies between exons) **only in eukaryotes Terminator: 3' end of the gene (termination) downstream of the gene Exon: coding Intron: non-coding
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14. Where is the poly A tail located? When and how is it synthesized?
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3' end of the mRNA. Polyadenylation of RNA: Important for translation and stability of RNA; polyA polymerase adds poly A tail to complete the mRNA synthesis
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14. Where is the cap located? When and how is it synthesized?
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5' end of the gene: added by capping enzymes; caps the end of pre-mRNA; aids in translation and prevents RNA degradation
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14. Which part of a primary RNA transcript contains the information encoding the amino acid sequence of a protein?
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Exons encode amino acid sequences
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14. Why is RNA able to perform the transesterification reactions required for splicing, but DNA cannot?
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RNA has a 2' OH but DNA does not
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14. What makes up snRNPs? What do they do? How do they recognize their targets?
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They are small nuclear ribonucleoproteins They recognize splicing sites and assemble a spliceosome that directs splicing ATP hydrolysis powers rearrangements that guide splicing
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14. When, with respect to initiation, elongation, and termination do each of the processing steps occur during the formation of a functional mRNA? What signals each step? What factors/enzymes does a pre-mRNA encounter during these processing events?
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1. 5' Capping by capping proteins during elongation When the RNA chain is ~30 nucleotides long, th
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15. What prevents the misprocessed or unmodified mRNAs from leaving the nucleus?
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Removal of introns?
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15. What RNA is most abundant RNA in the cell, and which RNA polymerase is responsible for synthesizing it?
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rRNA is the most abundant--> It forms the basic structure of the ribosome-->catalyzes protein synthesis RNA pol I is responsible for its synthesis
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15. What forms the nucleolus? What happens there?
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rRMA congregate in the nucleolus and it is where rRNA is synthesized
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15. Three different types of RNA are involved in translation. What are they and their functions? Which type contains codons and which type contains anticodons? Which type catalyzes peptide bond formation?
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mRNA: code for proteins Codon: 3 nucleotide units of mRNA tRNA: Central to protein synthesis as adapters between mRNA and amiono acids Anti-Codon rRNA: Catalyzes protein synthesis
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15. Why is the genetic code considered "degenerate?"
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b/c many different codons specify a single amino acid e.g. CUU, CUC, CUA, CUG all code for Leucine (L)
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15. Why is the reading frame important?
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If the codons are read wrong, then you make different polypeptide and possibly non-functional protein This is where synthesis starts, and it determines which amino acid starts and which amino acids make up the polypeptide The start codon sets the reading frame (AUG)
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15. What causes translation to stop at a stop codon?
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Stop codons: UAA, UAG, UGA When a stop codon is reached, instead of an amino acid, a termination factor comes and binds to this codon This causes the dislodging of the ribosome from the mRNA and translation is stopped
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15. What parts of an amino acid and the tRNA are involved in forming the covalent bond between these two molecules?
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tRNA: At the 3' end of the acceptor stem, there is a 3' OH group on the ribose sugar. Amino Acid: The carboxylic acid group The hydrolysis of these two forms a phosphodiester bond similar to that of the nucleic acid backbone.
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15. Which end of the polypeptide chain is encoded by nucleotides nearest the 5' end of a gene?
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The RNA polymerase begins synthesis at the 5' end of the mRNA. Because of this, the first amino acid to be translated is at the 5' end
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How does Aminoacyl-tRNA Synthase work? (5)
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Aminoacyl-tRNA Synthase binds ATP and AA
Adenosine-P-P-P lose 2Pi and its attached AA
Correct tRNA is bound and AA is transferred to tRNA
Adenosine monophosphate is released
Activated tRNA AA molecule is releasedhttp://highered.mcgraw-hill.com/novella/MixQuizProcessingServlet
aminoac…
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Structural features common to tRNAs
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Named for modified bases
DHU loop: dihydrouridine
TψC loop: ribothymidine, pseudouridine
Variable loop
AA acid attached to 3' end, phosphorylated 5' end
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15. Contrast rRNA cleavage and mRNA splicing.
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rRNA cleavage is a processing step for rRNA which uses snoRNAs and proteins both to do a substantial amount of bas modification and cleavage. This particular cleavage results in the separation of the 18S, 5.8S, and 28S ribosomal subunits mRNA splicing is a processing step for mRNA which u…
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16. Of what is the ribosome made?
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Large and small subunits; each of which has rRNA and multiple proteins 2/3 of the RNA and the rRNA forms the active site and tRNA forms binding sites
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16. What occupies the different tRNA binding sites?
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3 tRNA binding sites formed by the large and small subunits P-site: contains peptidyl tRNA and A site contains aminoacyl-tRNA E-site is the exit site
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16. What is meant by the terms poly and monocistronic?
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Poly: When an mRNA directs the synthesis of multiple proteins (bacteria) Mono: mRNA directs the synthesis of one protein (eukaryotes)
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16. How do prokaryotic and eukaryotic ribosomes initiate translation differently?
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P: Small subunit of the ribosome associates with specific sequences located adjacent to the start codons This allows ribosomes to initiate translation at multiple places on an mRNA E: Initiator-tRNA associates with the P-site of the small subunit The small subunit scans until the 1st AUG …
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16. What elongation factors are used in eukaryotic translation? What energy carrier do they hydrolyze?
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Ef-Tu: Escorts aa-tRNA into the A-site and helps to ensure a correct match to the codon in the mRNA and contributes to proofreading Ef-G: Stimulates
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16. Why is a ribosome called a ribozyme?
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an RNA molecule that is capable of catalyzing specific biochemical reactions, similar to the action of protein enzymes. The 1982 discovery of ribozymes demonstrated that RNA can be both genetic material (like DNA) and a biological catalyst (like protein enzymes), and contributed to the RN…
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What part of the ribosome serves the peptidyl transferase function?
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RNA
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How does a stop codon force translation to stop?
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It causes a release factor (protein) to bind the open A site, causing the nascent peptide to be released from the 3' end of tRNA at the P site.
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elF2
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protein synthesis initiation factor that brings the initiator met tRNA to the 30S ribosome prior to scanning (regulated by GCN2). binds to GTP to from complex. elf2-GRP is hydrolized to Elf2-gdp and elfs are releases
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16. What role does the 5' cap play in translation initiation? What recognizes the cap?
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cap binding protein helps the 40s ribosome with tRNA-met bind directly to the 5' end of mRNA. the ribosome the is able to slide up the mRNA chain and look for AUG. 5' cap is needed for efficient splicing of the intron nears the 5' end of pre-mRNA molecule
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Polyribosome
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a structure produced when many ribosomes simultaneously translate a single mRNA molecule . help to collectively synthesize protein
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16. What factors assist protein folding in the cell? What energy carrier do they hydrolyze?
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chaperones: HSP70: acts before protein is synthesized; HSP60 acts after by forming complex. finds hydrophobic segments. uses ATP hydrolysis
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Describe possible fates for a misfolded protein in a cell
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Molecular chaperones helped them to refold. Also, they are detected by "unfolded protein response". They are degradaded by the lysosomal pathway and by ubiquitin-mediated protein degradation.
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What is ubiquitin? What is it attached to?
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Ubiquitin are small proteins that are attached to misfolded proteins or old proteins that need to be degraded by the proteasome. Ubiquitin directs the targeted proteins to the proteasome.
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how are proteins targeted for degradation
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phosphorylation targets them for ubiquitination and destruction by proteosome
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phosphorylation targets them for ubiquitination and destruction by proteosome
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ubiquitin binds with cap
atp hydrolysis
confromational change of cap, causes a strain
strained ring structure pulls on substrate: ratcheting action
adp releases, new tap chain slips
cap: base with 6AAA family ATPase (gatekeeper: only allows protein with ubiquitin
core: contains enzy…
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17. What are the different points of control of gene expression? Which occur in the cytoplasm and which in the nucleus? Which is the main control point?
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...
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17. How do DNA binding proteins recognize specific sequences? Does this binding disrupt base pairing?
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amino acid ide chains h-bond to dan bases in the major groove; bind by contacting the edges of bases presented in MAJOR groove
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Why do most DNA Binding proteins recognize the major groove of DNA?
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The major grove is wide enough to fit an alpha helix
Functional Groups exposed at the edges of the bases are available for interactions with the protein s amino acids
Allow for discrimination of all 4 bases, (minor grove only G:C vs A:T
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17. What are the three most common types of DNA binding domains? What are their structures? What is the secondary structure that makes contact with DNA in all three types?
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Helix-turn-helix: Two alphas helices joined by a short string of amino acids. Zinc Finger: A small protein motif that contain multiple finger-like protrusions that make tandem contacts with their target molecule Leucine Zipper: Each half of a leucine zipper consists of a short alpha-helix…
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17. What type of amino acid is usually enriched in the DNA binding domain of proteins? Why?
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Typically positively charged amino acids are involved in the DNA binding domains because DNA is negatively bonded.
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What are the benefits of dimerization for DNA-binding proteins?
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a. it doubles the contact area with DNA; which increases the strength and specificity of the interaction
b. it makes it possible for many different DNA sequences to be recognized by a limited number of proteins
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17. What is the difference between bacterial operators and promoters? What proteins recognize these two sequence elements?
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core sequences recognized by the RNA polymerase holoenzyme; contains infer about where transcription will start and in what direction RNA polymerase will move. operator is a short region of DNA that is recognized by a repressor protein. a repressor is the protein that binds to the operato…
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17. Lambda repressor functions as a repressor at one promoter and an activator at another promoter in bacteria. How can the same protein have opposite activities at different promoters?
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if binding site overlaps the promoter, the polymerase cannot bind and protein acts as a repressor. it is location dependent.
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17. How does the tryptophan repressor regulate gene expression?
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allosterically activated by tryphtophan. when 2 triphtophan occupy it blocks access to promotor by RNA polymerase and prevents expression of triphtophan. without tryphtophan, motif swings inward and protein cannot bind
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lac genes are expressed coordinately through an operon (gene regulatory mechanisms for prokaryotes). regulated by a repressor and an activator (glucose and lactose).
lac operon cause use lactose as its energy. however, if there is glucose present, the lac operon will use that instead. tr…
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lac genes are expressed coordinately through an operon (gene regulatory mechanisms for prokaryotes). regulated by a repressor and an activator (glucose and lactose).
lac operon cause use lactose as its energy. however, if there is glucose present, the lac operon will use that instead. tr…
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18. What allows prokaryotic RNA polymerase to bind to multiple promoter sequences?
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The binding of RNA polymerase to the promoter is controlled by sigma factor. There are many different kinds of sigma factor, each of which can bind to a specific set of promoters. Because a single sigma factor can bind to more than one promoter, so too can RNA polymerase.
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18. What is the most significant feature that distinguishes the promoter recognition capacity of RNA polymerases in bacteria and eukaryotes?
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...
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18. How does a prokaryotic activator (or repressor) bind relative to the promoter? How does it differ from where a eukaryotic activator (or repressor) binds?
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prokaryotes: repressors bind to operators, located downstream from and near the promoter; activators bind upstream of promoter (cap region). eukaryotes use co-activators and co-repressors. use of enhancers, distant regions of DNA that can loop back to the promoter. ** eukaryotes have a nu…
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18. Why is it said that eukaryotic activators and repressors work "at a distance?"
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promote or block the binding polymerase, turning transcription of genes on and off. activator proteins are more important than repressors. default state for most genes is OFF. they don't need to bind to adjacent promoter. multiple regulators and GTFs bind DNA at promoters and regulatory r…
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18. How do eukaryotic activators increase transcription? How do eukaryotic repressors decrease transcription?
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increase: activators bind to the enhancer sequence far from the promoter; activators bend DNA to form a bridge b/w the GTFs assembled at the promoter and the activator. coactivator protein often connects the GTFs and activator protein. RNA pol II binds to DNA loop
decrease: inhibit activ…
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18. Mechanisms to control gene regulatory proteins? (7 methods)
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1) protein synthesis
2) ligand binding
3) protein phosphorylation
4) addition of second subunit
5) unmasking (use of inhibitor)
6) stimulation of nuclear entry (inhibitor)
7) release from membrane
{ 8) regulated proteolosis }
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what is the advantage of having introns that need to be eliminated before the mRNA can be translated?
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allows evolution to occur at increased pace
alternative splicing can occur, where one gene can be spliced in multiple ways to create different mature mRNAs
exons can be separated and rearranged to generate new genes and proteins of new function
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19. What is similar about the mechanism of regulation of alternative splicing in eukaryotes and transcriptional regulation in bacteria?
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exons can be arranged in alt ways when pre-RNA is spliced to make RNA to facilitate different levels of gene expression
transcriptional regulation in bacteria: genes arranged in operons where they are co-reguatled; change in gene expression levels
RNA polymerase actions; similar mecha…
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19. What are the 5' UTR and the 3' UTR? What are their functions?
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untranslated region; not used to make proteins; contains info about how long the sequin should be stable, how quick it can be translated. at both 5' and 3' of mature mRNA. made of micro RNA. region at beginning of mRNA. CONTAINS INFO ABOUT BINDING PROTEINS
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19. How are mRNAs destroyed? What determines if an mRNA is destroyed faster or slower? How are mRNAs destroyed? What
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miRNAs destroy mRNAs
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19. What are the mechanisms that regulate translation of specific mRNAs?
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Recruitment of the small ribosomal subunit can indeed be modulated by mRNA secondary structure, antisense RNA binding, or protein binding. In both prokaryotes and eukaryotes, a large number of RNA binding proteins exist, which often are directed to their target sequence by the secondary s…
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microRNAs (miRNAs)
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are small single stranded RNA molecules that bind to specific mRNAs and block translation or break them down. derived from primary miRNA hairpin transcripts. regulate gene expression; small single stranded RNA mole (complementary sequences of mRNA
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20. What is chromatography? Generally speaking, what does this technique allow an experimenter to do with a mixture of molecules?
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Chromatography is the collective term for a set of laboratory techniques for the separation of mixtures. It does so based on polarity
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20. What technique would you use to separate cell components, like organelles?
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centrifugation separates organelles and macromolecules. used to separate two immiscible liquids.More-dense components of the mixture migrate away from the axis of the centrifuge, while less-dense components of the mixture migrate towards the axis.
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20. What is gel electrophoresis? For what is it used? On what physical properties of the sample does it rely? On what physical properties of the gel does it rely?
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Definition: Method for the separation and analysis of macromolecules and their fragments based on their size and charge The porosity of the gel determines how quickly the substance(s) will move through the pores.
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20. What is gel electrophoresis? For what is it used? On what physical properties of the sample does it rely? On what physical properties of the gel does it rely?
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SDS-PAGE: Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis SDS-PAGE separates proteins based on size (i.e. #of amino acids) instead of intrinsic charge It is treated with Mercaptoethanol to break the disulfide bonds between the domains and SDS denatures the proteins and coats the…
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20. What is a Western blot? Why would you use one? What is the "probe" in a Western blot? Why is it used? Why is there a primary probe followed by a secondary probe?
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A Western blot is performed after SDS-PAGE and is used to isolate/identify a specific protein in the complex mixture. The probe is a specific antibody attuned to the protein in question. This primary antibody is directed against the particular antigen in question. The primary probe doesnt…
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20. How do you purify a protein? What techniques would you use?
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Protein purification involves successively passing fractions containing the protein activity over different columns in sequence Each step purifies the sample--as it removes more contaminating proteins Activity is determined by the experimenter - examples are ligand binding or catalyzed ch…
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20. What are some different types of column chromatography? What do they have in common? How are they different from each other?
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Ion Exchange: Gel Filtration: Affinity:
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20. What is mass spectrometry? What do you need to know in order to extract protein identity or sequence from this technique? and Why?
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fast and accurate way to measure MW. separates ions magnetically and determines ratio of charge to mass. allows for the identification of protein (provided the genome has been sequenced and a database has been compiled of the masses of predicted protein fragments). used to identify amino …
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X-ray diffraction & Atomic force microscope
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1 ways to determine crystal structure
X-ray diffraction: shoot a crystal with an x-ray, black spots are where there were atoms so flip the image and voila you have the structure
Atomic force microscope: like a gramophone, quartz crystal is piezoelectric (has an electric field), scrape t…
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17. Promoter vs Repressor vs Activator
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Promoter = site that RNA polymerase binds to to start mRNA formation; before operator which is blocked by repressor
Activator = bends DNA to start mRNA production (CAP)
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what is epigenetic inheritance
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a pattern in which modification occurs to a nuclear gene or chromosome that alters gene expression, but is not permanent over the course of many generations.
1. positive feedback of proteins
2. histone modification
3. DNA methylation
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B M B 251: EXAM 3