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94 Cards in this Set
- Front
- Back
Do eukaryotes have polycistronic or monocistronic mRNA? |
Monocistronic Each gene is regulated separately |
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Do eukaryotes have a specific repressor for each gene? Why? |
No Eukaryotes have many genes so a specific repressor for each gene is not practical |
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Per cell, approximately how many protein encoding genes are transcribed? |
10-20,000 |
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10-20,000 protein encoding genes are transcribed per eukaryotic cell. The majority of these are common (____________). Some are ________ specific. |
Housekeeping Tissue |
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What are housekeeping genes? Give some examples. |
Gene products are needed for general cell functions e.g metabolic enzymes (glycolysis pathway), cytoskeletal proteins (proteins for cell division) |
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What is an example of a tissue specific gene? |
Globin genes in developing RBC's |
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Different levels of regulation of gene expression are possible in eukaryotes. Where can gene expression be regulated? |
1. Transcription; initiation, elongation,termination 2. RNA processing 3. mRNA transport (out of nucleus) 4. RNA stability 5. Translation |
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What level of gene expression is most common and important? |
Transcription; initiation, elongation, termination |
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What is meant by RNA stability? |
How long mRNA hangs round for-determines how much protein you make |
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What is a promoter? |
DNA sequence that initiates and regulates transcription |
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Where can the promoter be found? |
Usually upstream (5') of the transcribed sequence |
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What are transcription factors? |
Regulatory proteins that recognise and bind specific DNA sequences Any protein that isn't RNA polymerase but assists in transcription |
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What does the arrow denote? What is the core promoter? |
Where transcription is going to occur core promoter = minimum sequence needed to get some transcription |
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What does RNA polymerase II do? |
Makes mRNA |
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What are these? |
Typical sequences we tend to find in most of regulatory promoter sequences |
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How do transcription factors bind to DNA? |
By specific amino acid chain:base interactions (N.B interaction = hydrogen bonding) |
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RNA polymerase II requires general transcription factors. What are some things that these transcription factors should do? |
Bind to all promoters Position RNA polymerase at the promoter Help to separate DNA strands to allow transcription to being Allow RNA polymerase to leave promoter as transcription begins (prevents RNA polymerase getting stuck) |
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Just one interaction on its own wouldn't achieve any sequence specificity-the DNA binding protein could just bind anywhere is there was an A-T base pairs. If next to that amino acid there was another amino acid that bound to a different b.p sequence, etc, then you would build up ___________ __________ |
sequence specificity |
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What is TBP? |
TATA binding protein An example of a general transcription factor |
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What complex is TBP part of? |
TFIID complex |
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What protein binds TATA box? |
TATA binding protein |
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TBP is a "_____________ factor" needed for transcription of mRNA encoding genes |
commitment |
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Where does TBP bind to DNA and what does it do once it has bound here? |
Binds DNA in minor groove and bends it, this opens up the DNA and allows other proteins, including RNA polymerase, to recognise and bind promoter |
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TBP is needed for transcription. Is it enough on its own? |
No |
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What is required for RNA polymerase II to being transcription? |
General transcription factors |
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How are transcription factors named? |
TF = transcription factor II = related to RNA polymerase II A/B/D related to order of discovery |
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What do you know about TFIIH? |
Acts with DNA helicase Has kinase activity, so phosphorylates part of RNA polymerase so it can leave promoter and be mobile and move along down sequence |
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How do TF's activate (or repress) transcription? |
Interacting with pol II and/or general transcription factors Modification of chromatin |
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Where are transcription factors typically found? |
~ 100 bases upstream of transcription site |
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What are enhancers? |
DNA sequences that regulate eukaryotic gene expression (N.B can even be within an intron) |
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Normally, how long are enhancers? |
200 bp long |
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Where do enhancers function in respect to a promoter? |
Up to 50 kbp upstream of downstream of promoter |
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What do enhancers contain? |
Multiple binding sites for transcription factors |
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Enhancer-bound transcription factors alsointeract with the ______ _____________ ________,and/or modify ________. |
basal transcription complex chromatin |
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What is a mediator? |
A complex of proteins that links upstream-bound transcription factors to basal complex |
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What is chromatin? |
The material (DNA and protein) of chromosomes |
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What are the major structural proteins of eukaryotic chromosomes? |
Histones |
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Histone are DNA-binding proteins. Approximately, what is their Mr and how many amino acids are they composed of? |
Mr 20-30,000 100-135 amino acids |
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Histones contain a high proportion of what kind of amino acids ( gives 2 examples) and why? |
Basic amino acids ( positive charge) E.g Lysine and arginine Helps binding to negative DNA |
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What are the 5 histone proteins? |
H1, H2A, H2B, H3, H4 |
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What do we know about H2A and H2B? |
They have closely related sequences |
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What is a nucleosome? |
The basic subunit of chromatin 2 molecules each of H2A, H2B, H3 and H4 (the histone octamer) with 146 bp of DNA wrapped round them (1.8 turns), plus linker DNA each side |
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What is the total DNA per nucleosome? (N.B this is DNA wrapped around each nucleosome plus linker DNA) |
200 bp |
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What is the diameter of a nucleosome? What is the diameter of DNA? |
10-11nm 2 nm |
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What does this show? |
Chromatin in a low salt solution outside of cell |
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What does histone 1 (H1) do? |
Binds DNA where it leaves and enters the nucleosome core |
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What does Histone H1 aid? |
Further packaging of nucleosomes into a 30nm fibre (i.e helps chromatin to wrap up further) This is thought to have the form of a solenoid (coil) |
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The human chromosome in the 30 nm fibre would be 0.1 cm long. Therefore further packaging is needed. How is this achieved? |
The 30 nm fibres are folded into loops that are attached to a scaffold of non histone proteins |
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What happens at M phase? The net result is that each DNA molecules has been packaged into a mitotic chromosome that is _________ shorter than its extended length |
Looped chromatin is wound up further 50,000 x |
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Chromatin is a dynamic structure. How can transcription factors access and bind DNA? |
'Chromatin remodelling' Looser packing |
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What is meant by histone acetylation? |
An acetyl group can be added to the lysine/arginine group to make it lose its positive charge so it binds less tightly to DNA |
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What is HAT? What is HDAC? |
Histone acetyl transferase Histone deacetylase |
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Histone acetylation reduces __________ charge so histonetails bind DNA ____ tightly, allowing further __________ ________and ___ __________ to bindDNA. |
positive less transcription factors RNA polymerase |
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Tissue specific transcription factors bind to the promoter and enhancer sequences leads to what? |
Tissue specific gene expression |
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What transcription factor is specific to developing muscle cells? |
Mao D |
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The transcription factor MyoD is specific to developing muscle cells. What sequences does it bind to in the promoters and enhancers of muscle specific genes? |
'E box sequences' |
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Lab cultured fibroblasts + MyoD ------> ? |
Muscle cells |
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What are transcription factors? |
Proteins that bind to regulatory sequences in genes |
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What does moD drive the expression of? |
Gene that we need to develop muscle |
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What are some muscle specific genes that respond to MyoD? |
Muscle actin Creatine kinase |
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Where does the muscle Muscle Creatine Kinase (mMCK) gene contain E boxes? |
In its promoter and enhancer sequences N.B this sequence will give muscle specific expression |
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What is the E box sequence? |
CANNTG N.B N can be any one of the 4 bases |
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E-box sequences and promoter sequences are responsible for driving what? |
Muscle specific expression |
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We can move the regulatory sequence from the mouse genome to a different type of embryo. The enhancer and promoter sequences are bound by MyoD and drive muscle specific expression of what? |
GFP |
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What does this show? |
Not only do developing muscle cells start to express these muscle specific genes, but also as cell divide, the progeny of these cells retain the ability to express muscle specific genes. |
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How can tissue specific gene expression be maintained when DNA replicates and cells divide? |
DNA methylation |
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Which base in mammalian DNA is extensively methylated? |
Cytosine |
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What is a consequence of having methyl groups on cytosine? |
A gene will not be expressed |
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Methylcytosine is associated with what? |
Transcriptional inactivation |
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Why does methylation of cytosine prevent transcription of a gene? |
Methylation may prevent transcription factors from binding at promoters Methylated DNA can be bound by specific proteins that form complexes with histone deactylases N.B histone deacetylases are enzymes that modify chromatin and shut it down and make it not be expressed as it is bound so tightly to histone protein that transcription factors cannot access promoter |
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Methylation allows stability of cell lineage. DNA methyltransferase enzymes recognise methylated C in template strand and so what do they do in the replicated strand? |
Methylate C |
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Eukaryotic DNA methylation is a different phenomenon from prokaryotic DNA methylation. How? |
Prokaryotic DNA methylation 'marks' the template strand to allow DNA repair and protects cell's own DNA from digestion by restriction enzymes |
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Gene expression is regulated in response to signals from outside the cell. Describe how steroid hormone receptors can act as transcription factors. |
Hormone bound receptors binds to DNA Receptor sits in cytosol until hormone enters cell. When hormone binds to the receptors, its conformation changes so it moves into the nucleus. The receptor recognises and binds specific DNA sequences and activates their expression in response to the hormone (cortisol). |
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Many mammalian genes are transcriptionally activated by cAMP. What do they therefore have in there promoter sequence? |
The cAMP-responsive element N.B this is like the Ebox in muscle promoters that binds to myo D |
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What is a transcription factor (a cAMP-responsive element binding protein) that is activated by cAMP? |
CREB |
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What is an example of a cAMP responsive gene? |
Carboxykinase |
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What does PEP catalyse the formation of? |
Phosphoenol pyruvate from oxaloacetate in gluconeogenesis |
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CREB is a transcription factor activated by hormone signalling via protein kinase A. Describe this sequence of events. |
N.B example hormone is adrenaline but can also be glucagon (with a glucagon receptor) |
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What does the phosphorylate CREB help to do? |
Recruit RNA polymerase II and histone acetyl transferase to its target genes |
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In response to a decrease in blood glucose, there is increased glycogen degradation, decreased glycogen synthesis and increase gluconeogenesis. Deci |
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Which of these is the slowest response and why? |
Increase gluconeogenesis You are making enzymes rather than activating them |
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What are the different levels of regulation of gene expression in eukaryotes? |
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RNA interference (RNAi) regulates eukaryotic gene expression at the post-transcription level. What can the regulatory RNAs be? |
Exogenous in origin (small interfering RNAs or siRNAs) e.g from viruses or transposons Endogenous in origin (micro RNAs or miRNAs) >1% of human genome Aprox. 1/3 of all human genes are targeted |
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What are siRNAs produced from? |
Double-stranded foreign RNAs as a defence mechanism |
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Describe how siRNAs are produced from double stranded RNA |
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What happens when the foreign double stranded siRNA complexes with RISC protein? |
One strand is removed (passenger strand) and the other one remains as the guide strand. The guide strand+RISC proteins go looking for cDNA. |
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What is a dicer? |
A nuclease enzyme that chops RNA into smaller bits |
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What does RISC stand for? |
RNA-induced silencing complex |
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Describe what happens to each of the strands of RNA when it interacts with the RISC protein. |
One strand ( the passenger stand) of the siRNA is removed by ribonuclease digestion The remaining guide strand binds to its complementary target RNA |
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miRNAs target complementary mRNAs for destruction as part of normal gene regulation. Describe how. |
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What happens when the RISC complex finds an extensive match? |
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What happens when the RISC complex finds a less extensive match? |
Less exact match prevents ribosome from being able to translate RNA efficiently Eventually RNA is broken down as it is not being translated |
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miRNAs could be used for therapies therapeutically and experimentally e.g in cancer cells to stop them expressing particular genes. True or false. |
True |
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What are miRNAs transcribed from? |
Part of our genome that doesn't encode proteins |