BIOL253 L13

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  • Created by: Katherine
  • Created on: 11-04-17 19:02
Why do cells need to regulate transcription?
While a cell might require a lot of some proteins, they might only require a little of others. They also need to produce some proteins at particular times .
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What is a cell type that is only needed under certain conditions?
Alternate sigma factors which are requires under stressful conditions
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RNA polymerase holoenzyme can transcribe any gene with a...
Functional promoter
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Targeted gene regulaton is used most when?
At transcription initiation (most often), at elongation or termination and during regulation from the transcribed RNA itself.
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Regulatory proteins that decrease transcription are called...
Repressors.
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Proteins that increase transcription are...
Activators
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The regulatory DNA sequences that regulatory proteins bind to are called
operators (operons)
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It is far more common for a eukaryotic gene to be regulated by multiple proteins. In multicellular organisms, regulatory sequences can encompass binding sites for 6 or more proteins and may be located at great distances. What are these called?
Enhancers,
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What are enhancers?
They bind proteins that activate transcription.
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Where are enhancers located?
An enhancer can be located either upstream or downstream of the target gene. The region of the chromosome over which an enhancer acts can be delimited by an insulator sequence.
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How do enhancers regulate transcription?
They work at distant promoters because the DNA forms a loop that allows proteins bound to the enhancer region to contact proteins bound to the promoter region.
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What are locus control regions?
Complex regulatory regions which contain a combination of enhancer and insulator elements.
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Give an example of something controlled by a locus control region:
Beta globin genes
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How do LCRs work?
Preinitiation complex forms, binding of tissue specific factors. Gene specific activation by LCR promoter conact. Model for looping between LCR and promoter region.
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Give an example of binding domains found in TFs:
Helix turn helix (found in all types of organisms) and Zinc finger domains which are found in Humans. And Coiled coil domains (found in leucine zipper and helix loop helix motifs)
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How are constitutive or housekeeping genes expressed?
Constitutively expressed - encode proteins cell always needs e.g for glucose metabolism.
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How are regulated genes expressed?
Expression required only under certain circumstances e.g. changes in temp or availability of nutrients. Expression is regulated at the level of transcription.
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Give an example of a regulated gene and explain
LexA repressed expression of SOS genes in the absence of dmage. LexA binds to the operator sequence and stops RNA polymerase from transcribing the SOS genes. When the cell encounters DNA damage, the repliction fork stalls.
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What happens when the replication fork stalls?
RecA binds to single stranded DNA and becomes activated to cleave the Lex A repressor. Lex A cannot bind to DNA and so there is transcription of SOS proteins including red A and Dinl
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What happens once the DNA damage is repaired?
RecA is inhibited by Dinl and newly synthesised LexA repressor binds to DNA.
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What is an operon?
A portion of DNA that has genes with proteins which work in a co-ordinated way.
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What are operons responsible for?
Expression of genes which encode proteins that work together (e.g. to metabolise lactose or to synthesise tryptophase).
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In an operon, genes adjacent to each other are...
Transcribed together into one polycistronic mRNA. Which is then translated into separate proteins encoded by each gene in the operon.
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What is the main source of energy in E.coli?
Glucose
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What happens when lactose becomes the sole carbon source?
Rapid increase in lac mRNA transcript (transcript is unstable). Rapid synthesis of enzymes required to metabolise lactose.
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What is the function of B galactosidase
It converts lactose to galactose and glucose or to allolactose.
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What is the role of permease?
To import lactose to the cell
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What is LacL
This produces the lac repressor protein - the lac repressor has its own promoter which is constitutively active. The E.coli is contiunually producing the lacI protein.
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Why is the operon (LacZ,lacY and lacA) produced?
To produce three proteins: B galactosidase (LacZ), Permease (lacY) and Transacetylase (LlacA)
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What happens when there is no lactose present?
When there is no lactose present, LacI binds to lacO (the lac operator). No transcription occurs as RNA Pol can't bind
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What happens when lactose is present?
Allolactose binds to LacI, altering its shape. This allows transcription to occur (derepression). This is because the lac operon is no longer able to bind the lac repressor.
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What happens when glucose is present?
Presence of glucose inactivates adenylate cyclase and dramatically reduces levels of cAMP.
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cAMP is an indicator of...
Glucose level. cAMP high = low glucose, cAMP low = high glucose
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What is CAP
Catabolite activator protein . Only binds to DNA in the presence of cAMP. It bends the structure of the DNA by 90'c.
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When does CAP bind and what does it do?
CAP -cAMP binding is upstream of the promoter - it facilitates RNA polymerase holoenzyme binding.
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Where does the cell get allolacose from?
Through the activity of betagalactosidase
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The lac operon is regulated by a combination of..
Negative inducible regulation (lac repressor) and positive regulation (via cAMP-CAP)
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What is negative regulation y repression?
Operons for anabolic pathways turned off when end product is readily available e.g. try operon.
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Expain negative regulation in the trp operon:
5 Gene encoding enzymes for tryptophan biosynthesis are expressed in an operon as a polycistronic mRNA - turned off by excess tryptophan
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What does the Trp repressor do?
It blocks RNA polymerase binding
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When cellular lebels of tryptophan are low what happens?
Genes that encode enzymes required for the synthesis of tryptophan are expressed.
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When levels of tryptophan are high, what happens?
Transcription from the trp operon is repressed. This is accomplished by the Trp repressor, which binds to a site directly overlapping the promoter and thereby blocks access by RNA polymerase.
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TrpR encodes an...
Aporepressor
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In the absence of tryptophan,
no binding of the TrpR aporepressor to operator - transcription occurs
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In the presence of excess tryptophan,
aporepressor activated by binding tryptophan (acts *** co-repressor_. Binds to operator, preventing transcription.
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What is tryptophan starbation?
f
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Four regions of the trp leader mRNA can form..
Alternative secondaty structures
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In low tryptophan presence...
There is an incomplete leader peptide. An antitermination loop is formed.
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In igh tryptophan,...
There is a completed leader peptide.
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What are the key features of trp operon regulation
On/off switch by negative repressible reguation. Absence of tryptophan = no repression = transcription. Excess tryptophan = Trp R aporepressor + tryptophan co repressor bind operator - no transcription.
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What are roboswitches?
Portions of a transcript that can directly bind a small molecule that controls the RNA secondary strcture, regulating transcription or translation.
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How mant regions do riboswitches have?
2 - The aptamer that binds to the metabolite and an expression platform which control transcription or translation
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What doees the adenine riboswitch do?
It regulates adenine synthesis and transport. Gene expression depends on whether a terminator or anti-terminator forms.
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In low adenine, what happens?
The RNA structure haas an anti-terminator formed and transcription proceeds.
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In high adenine, what happens?
Regions 3 and 4 form a terminator to stop transcription.
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