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FRNSC 420 - EXAM 3-Questions with Correct Answers/ Verified/ 100% Pass
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FRNSC 420 - EXAM 3-Questions with Correct Answers/ Verified/ 100% Pass
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FRNSC 420 - EXAM 3-Questions with Correct Answers/ Verified/ 100% Passelectrophoretic mobility shift assay (EMSA) - ✔️✔️demonstrates if a protein is bound to DNA
- fragment is placed in non-denaturing polyacrylamide gel (native PAG) to keep protein associated to
DNA
- DNA/protein complex moves much slower in the gel due to added molecular weight (an
electrophoretic mobility shift)
- will appear higher in the lane compared to free DNA
- migration depends on mobility of the protein; different protein/DNA complexes migrate at
different rates
consequences of transcription - ✔️✔️expression products of genes that have specific functions
- phenotypic function (ex: vision)
- cellular function/processes (ex: metabolism)
- molecular function (ex: regulation)
transcriptome - ✔️✔️genes that are expressed in a certain cell type under a given set of
environmental conditions
prokaryotic RNA polymerase - ✔️✔️core subunits are β, β', α', α'', and ω
- initiation of transcription at promoter sequences facilitated by addition of σ factor to core
polymerase
eukaryotic RNA polymerase - ✔️✔️three different polymerases, each with their respective five
subunits
- Pol II: most active, transcribes most genes
- Pol I: transcribes larger rRNA precursors
- Pol III: transcribes tRNA genes, small mRNAs, and 5S rRNA gene
DNA foot-printing - ✔️✔️demonstrates if and where a protein is bound to DNA
- protein acts as a barrier against cleavage (from either DNase or reagents of the assay); DNA outside
the DNA/protein region is susceptible to cleavage
- denaturing polyacrylamide gel (Urea-PAGE) gel to keep the protein separated from the unbound
DNA; allows the protein to protect only the DNA its bound to & prevent it from affecting the DNA
mobility
, - nuclease will cleave DNA into random fragments in unpredictable patterns, avoiding the
DNA/protein complex
- a 'footprint' will appear in gel with a region of no bands within a lane of fragments
- having a control lane allows for visualization of where the protein may be bound
DNA replication vs RNA transcription - ✔️✔️- dNTPs in replication, rNTPs in transcription
- RNAPs initiate transcription without a primer, but can only transcribe a subset of the DNA template
- product is ssRNA
- multiple transcription events can occur sequentially from the same gene
- RNA transcription is less accurate than DNA replication
RNAP proofreading - ✔️✔️lowered RNAP infidelity may affect gene expression and protein function
- pyrophosphorolytic editing: enzyme uses its active site to catalyze removal of incorrect rNTP
through reincorporation of PPi
- hydrolytic editing: backtracks and cleaves RNA product by exonuclease-like hydrolysis, stimulated
by Gre factors
describe the structure, important features, and nomenclature of the region of target DNA around
the site of transcription initiation - ✔️✔️transcription begins at the +1 transcription start site (TSS)
- upstream (before) the TSS does not get transcribed; includes proximal (promoter/operator)
elements that aid in initiating transcription
- upstream is negative because it's moving against the direction of transcription
- downstream region moves in a positive direction (same direction as transcription) and is
transcribed
consensus promoter sequence - ✔️✔️promoter sequences are highly conserved 'landing zones' for
RNAP to bind
- most conserved sequences are the -35 bp and the -10 bp sequences
- defines the binding affinity and strength RNAP has to the sequence
- RNAP is specific, may not bind as well to discrepancies in consensus sequence
- closer the promoter is to the consensus sequences, the stronger the association of the holoenzyme
to the binding sites
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