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What two methods of transcription termination is present in prokaryotes?
- Rho-dependent termination
- Rho-independent termination
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Why might RNA pol pause or backtrack?
- Pausing (1-6 sec in vitro) is critical for two reasons:
- it allows a slower process of translation to keep up with transcription (and transcription will abort altogether if translation fails!)
- it is also a prerequisite of transcription termination
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In vitro termination assay
[How can one study the functionality of potential terminators (aka attenuators) in vitro?]
The size of the transcript will change dependent on whether the attenuator works -> differential electrophoretic mobility
One can then change the sequence of the terminator (mutagenize) and test the ability of RNA Pol to terminate
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constitutive expression
genes that are required at all times, such as 'housekeeping' genes
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What is an alternate sigma factor usage?
- E. coli often use alternative σ factors to respond to changes in environmental or nutritional conditions
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What class of factors (and holoenzyme subunits) do most DNA-binding transcription factors interact with?
- α (class 1 factors)
- σ (class 2 factors)
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Which holoenzyme subunits do factors (and related classes) interact with in the absence of DNA?
- β (class III factors)
- β' (class IV factors)
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Positive Regulation
(activation): a positive regulatory factor (activator) improves the ability of RNA Pol to bind to and initiate transcription at a weak promoter
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Negative Regulation
(repression): a negative regulatory factor (repressor) blocks the ability of RNA Pol to bind to and initiate transcription at a strong promoter
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Example of negative regulation in the lac operon
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Example of positive regulation in the lac operon:
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Describe how DNA looping can result in positive transcription regulation:
- A DNA loop can be responsible for transcriptional repression. A bivalent transcription factor binds simultaneously to two binding sites and blocks access to the RNA polymerase (e.g., the regulation of the lac operon, the best studied in E. coli).
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Describe how DNA looping can result in negative transcription regulation:
- A DNA loop can be responsible for transcriptional activation. Transcription factors bind away from the site of fixation of RNA polymerase (normally of σ54 type) and help the recruitment of RNA polymerase and formation of an open complex (in E. coli, the glnALG operon is regulated by this mechanism).
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operon
grouping of genes that encode proteins with related functions
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What are the three functional parts of an operon?
- structural genes: these encode proteins (usually with related functions)
- promoter: recruits RNA polymerase
- regulatory sequences: interact with regulatory proteins
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What is the lac operon's purpose?
E.coli prefers glucose, but will process lactose in the presence of lactose and the elimination of glucose
The genes responsible for the assimilation of lactose are organized in a single lac operon that is activated in the presence of lactose only when glucose in the medium is used up
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What do the regulatory regions of the lac operon include?
- CAP binding site
- promoter
- operator
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What structural genes are located in the coding region of the lac operon?
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lac Z purpose
lacZ encodes b-galactosidase, an enzyme that cleaves lactose into galactose and glucose, both of which can be used by the cell as energy sources
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lac Y purpose
lacY encodes lactose permease, a 46 kDa membrane-bound protein required for importing b-galactosides (including lactose) from the media into the cell
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lac A purpose
lacA codes for transacetylase, a 23 kDa protein that detoxifies thiogalactosides taken up by the permease
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Which gene is responsible for regulating transcription of the lac operon?
lacI
Transcription of the lac operon is regulated by a 154 kDa tetrameric Lac repressor protein encoded by a separate gene, called lacI, constitutively expressed from the PI promoter
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When does the lac repressor bind to the operator site in the lac operon?
In the absence of lactose, Lac repressor binds to the operator site of the lac operon
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How do you induce the lac operon
it is induced in the presence of lactose/allolactose
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What is a synthetic inducer for the lac operon?
- IPTG
- IPTG>allolactose>lactose
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CAP
- catabolic activator protein
- binds up straeam of the lac operon promoter (at the CAP site)
- allosterically regulated by cAMP
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What is the regulatory mechanism that represses other sugar metabolism as long as glucose is present?
E. coli have developed a regulatory mechanism, called catabolite repression, that prevents the expression of genes for lactose, arabinose, and other sugars in the presence of glucose, even when these secondary sugars are also present
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In which gene mutants can bacteria no longer process lactose?
- Bacteria with mutations in either the lacZ gene or the lacY gene can no longer metabolize lactose:
- the lacZ mutants [lacZ- strains] because b-galactosidase activity is absent
- the lacY mutants because lactose is no longer transported into the cell
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How can one genetically distinguish between the repressor I- mutations and the operator Oc mutations, both of which constitutively express the lac operon genes?
By using merodiploids, i.e., partially diploid, E. coli strains that have a second wild-type copy of the lac operon introduced in its genome (or on a plasmid)
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Transcription
synthesis of RNA, using DNA as a template
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Name of the DNA strand that RNA is being written from
Template strand (non-coding strand)
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Name of the DNA strand that ultimately reflects the amino acids created when the RNA strand is read
coding strand
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Where is the promoter in bacteria?
Everything upstream of the +1 site
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In eukaryotes, where is the promotor?
spans the +1 site
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What did the experiment by Hinkle and Chamberlin conclude?
the sigma factor is important for RNA Pol binding to DNA
- the holoenzyme would stick for hours, but the core quickly disassociated
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What is the size of the initiation complex (with sigma) vs the general elongation complex (without sigma)?
- Initiation complex ~75bp
- general elongation complex ~30-40 bp
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What sigma factor targets 'housekeeping genes' for transcription?
sigma 70
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In CRISPR editing of a human cell line, if you wanted to favor HR over NHEJ, what can you do?
knock-down the genes for NHEJ in your cell culture
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Which part of the cell cycle is unlikely to repair damaged DNA via HR?
not before the DNA has replicated in G2
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If you wanted E.coli to express its heat shock genes constitutionally (irrespective of heat treatment) what would you need to do?
change the promoter to recognize the sigma factor that is used to respond to heat shock
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