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Transcription makes mRNA. mRNA can be processed (ie. capped and spliced). Stability is based on the rRNAs ________ _______.
Transcriptional regulation can be _____________ or ______________.
A repressor-binding site overlaps the promoter. When the repressor protein binds, RNA polymerase cannot initiate transcription and no mRNA is produced.
promoter and recruits RNA polymerase to initiate transcription.
In terms of activating transcription, why would DNA looping occur and how does this affect the regulation of transcription?
The regulatory site is far away from the promoter.
A transcription activator (blue) binds the regulatory site, promoter and RNA Pol forming a DNA loop .
Transcription is activated.
The bacterial Lac repressor protein, a tetramer of identical subunits, binds two distant sites on a single DNA molecule, forming a DNA loop.
DNA looping can be mediated by a single transcription factor. Give an example.
In the case of eukaryotes, the regulation could be far away (up to ___ _____ away from the promoter). So the DNA has to be looped around.
Certain transcription factors play an _________________ role in regulation.
They bend the DNA when they bind their DNA site, thus promoting looping.
The regulator facilitates looping for recruitment of RNA polymerase by an upstream activator.
Coactivators and corepressors bind regulatory proteins without
making direct contact with DNA. They make conformational change to the regulatory proteins to change their DNA binding ability.
This either stimulates of lowers their function and activity.
Coactivators bind transcription activators and facilitate their function in activating RNA polymerase.
In the case of the lac operon, we will look at the one example where the ________________ (co-activator or co-repressor) has to kick in.
Corepressors bind transcription activators and inactivate their
Eukaryotic promoters have _______ (many/few) regulatory elements.
Sequences that interact with insulator proteins to block the
action of activators on the promoter of a given.
When the regulatory sites for gene A are filled, the activators act on the promoter of gene A, but the insulator sequence binds insulators which blocks their action on the promoter of gene B.
Insulators are involved in blocking the activity of ______________ _______________.
How many activators to prokaryotes need for transcription to occur?
That large number of activators in different combinations can affect the _________ ________________.
Insulators bind their own regulator regions and protect a gene from being ____________________.
The regulation of gene expression usually operates as a _______________ _______________.
The effector changes the ______________ of the repressor or activator.
binds an activator or repressor and modulates its activity.
Effectors are signal molecules that act on repressors to relieve or enhance repression.
The binding of effectors to activators can inhibit or enhance activation.
(a) Activator (blue) binds in the absence of the effector and transcription proceeds; effector binds and inactivates the activator to inhibit transcription
(b) The effector binds and activates the “Activator (green)” to stimulate transcription.
Sets of genes may be regulated together in both prokaryotes (most often bacteria)and eukaryotes (eukaryotes we rarely have that).
What types of genes would typically be grouped together and what is the primary transcript produced from these genes called?
In bacteria often groups of genes “involved in a common pathway” are transcribed together. These are called an operon.
Transcription produces a polycistronic mRNA.
Some genes are regulated in a coordinated manner.
e.g. regulation of a bacterial regulon.
Some chloroplast DNA is very much like bacterial DNA and gives situations like that. But in typical eukaryotic DNA you have one regulator for one gene.
In polycistronic genes, how are the genes oriented and what is the significance of the proteins that are encoded?
The activator is produced and turns all the genes on at the same time.
If the genes are all repressed usually, somehow the cell decided to turn all the genes on in which case it attacks the repressor and all the genes get turned on.
are typically near, or overlap, the promoter.
are usually regulated by only one or two transcription factors
In prokaryotes, there is often _____ promoter region.
In eukaryotes promoter is within _________ nucleotides (usually ___bp) of the start site.
Multiple Regulators Provide Combinatorial Control of gene expression in eukaryotes.
how multiple Regulators Provide Combinatorial Control of gene expression in eukaryotes.
Each promoter requires five different regulatory proteins.
Each gene uses different combinations of transcription factors
Some factors are used for more than one gene.
Gene activation and repression in both bacteria and eukaryotes require ________________ _______________.
Proteins that affects the regulation and transcription initiation of a gene by binding to a regulatory sequence near or within the gene and interacting with RNA polymerase and/or other transcription factors.
TFs interact with DNA and proteins through ______________ _________.
The recognition of DNA by a TF is almost always through certain
amino acid side chains of an α helix referred to as the recognition helix.
What is the general state of gene expression in prokaryotes?
What does this involve? (3)
Prokaryotic genes are mostly constitutively expressed.
RNA Pol generally has access to every promoter
Most bacterial genes are controlled by specific repressors.
Regulatory molecules stimulate or inhibit RNA Pol binding to the promoter
What is the general state of gene expression in eukaryotes?
order to be transcribed.
order to be transcribed. Why?
is inaccessible to RNA Pol.
Open structure: Nucleosomes are often acetylated, and accessible to RNA Pol
Closed structure: Nucleosomes are methylated, and inaccessible to RNA Pol
TFs are classified based on the presence of specific conserved motifs.
TFs are classified based on the presence of specific conserved motifs. What are the different conserved motifs that transcription factors take on? (5)
The helix-turn-helix DNA-binding motif
The homeodomain DNA-binding motif
The leucine zipper motif
The basic helix-loop-helix motif
The zinc finger motif
The helix-turn-helix DNA-binding motif.
General characteristics. (4)
The helix-turn-helix DNA-binding motif.
The DNA-binding domain of the bacterial Lac repressor (as a ribbon structure) interacting with the major groove of DNA. The helix-turn-helix motif is red and green; the DNA recognition helix is red.
DNA binding site and an protein binding site (activating site).
What is a motif?
Any time a protein forms a secondary structure is a motif.
Any part of the protein that is made up of a number of motifs that can fold upon itself forms the ______________.
The domain is a portion of the transcription factor (or any protein) that has a function on its own.
The DNA recognition site of the transcription factor recognizes a ___________________ sequence and binds to it.
This is one large circular chromatin and it is not as densely packed. This is different from eukaryotic chromatins because they are very tightly packed so the RNAP II cannot get in there as easily.
The DNA is wound around histones which can be acetylated or methylated. It can be open or closed, which allows or disallows RNAP (ie. the DNA is either accessible or inaccessible).
If it needed to express all the proteins at once, this would cost the cell a lot of time and energy.
The theory is that the different state of expression in prokaryotes and eukaryotes has to do with the _________ and _________________ _________ of the genome.
defining body part formation (ie. flower development, body segment development).
Only one of the helixes interacts with the DNA. The other interacts with the dimer.
because the major groove, as you can see, is much more open. A bigger molecule can fit in there.
The functional groups on the amino acids are open for hydrogen bonding with the TFs.
In the ___________ groove, there is no space for the RNAP and TFs.
Binding of transcription factors is _____________ (reversible/irreversible).
The motif is generally not very stable but once it binds and dimerizes then it becomes really stable.
Homeodomain is found in proteins from a wide variety of multicellular organisms, including humans.
It contains a unique helix-turn-helix motif.
The homeodomain is composed of three α- helices, only two of which (helices 2 and 3) correspond to the helix-turn-helix motif
The N terminal residues of the homeodomain reach around the DNA and interact with the minor groove.
The Drosophila transcription factor
known as Paired. The recognition helic in each subunit is stacked on two other α helices and can be seen protruding into the major groove. The N-terminal sequence inserts into the minor groove.
Homeodomain TFs are typically found in ______________ (prokaryotes/eukaryotes) and affect development in terms of...
These have a unique helix-turn-helix in that there are three alpha helixes, each which correspond to the helix turn motif.
The basic leucine zipper motif is an _______________ __ helix
It is found in many eukaryotic and a few bacterial regulators
A striking feature of thisα helix is the occurrence of Leu residues at every seventh position, forming a hydrophobic surface along one side of the helix, where two identical subunits dimerize.
Many regulatory proteins that use leucine zipper helices only for dimerization and contain a separate motif for DNA binding.
Molecules that have both a hydrophobic an hydrophilic end. Because of the folding pattern in folding, these molecules have hydrophobic amino acids on one site and hydrophilic amino acids on the other site.
There are these Leu residues at the every seventh position. These Leu sites` that are highly conserved. Because of the folding structure, they are what creates the hydrophobic region.
You can put a number of the Leu zippers on a DNA molecule. What will be the result?
They are called basic because there are basic residues at the end (K and R).
mediate protein-protein interactions in eukaryotic transcription factors.
A zinc finger domain consists of about 30 residues that form an elongated loop held together at the base by a 1 (in type I) or 2 (in type 2) Zn2+ ions.
The Zn2+ ion is coordinated to four amino acid side chains, usually four Cys, or two Cys and two His residues.
Zn2+ stabilizes the motif, which presents a recognition helix to DNA.
Many DNA-binding proteins have multiple zinc fingers. What is the effect of this on the DNA?
The Zif268 recognition helices enter the major groove of the DNA, and the three fingers wind around the DNA helix.
The key feature here is that the DNA is stabilized by zinc.
A second type of zinc finger protein combines the Zn2+-binding motif with the helix-turn-helix motif.
These proteins bind to the DNA as dimers, using a leucine zipper to mediate the dimer contacts.
Zinc finger motifs are common in ________________, and there are a few examples among _______________ regulators.
Fill in with prokaryotes/eukaryotes.
The Gal4 protein (Gal4p), a yeast transcription activator, is a dimer, held together by a leucine zipper.
In this case, there are two complexes which dimerization and a new zipper region is formed between the two. This uses two Zn molecules. The dimerization is formed via a Leu zipper region. This is usually in eukaryotes. This is the basis of many systems (ie. the two-hybrid yeast system).
The binding domain is separated from the activating domain.
the DNA binding domain of TFs is separated from its transcription domain?
Construct a DNA fragment in which the lacZ gene so you can use blue-white selection.. Fuse the gene to the promoter of a LexA gene. Take the DNA binding motif of LexA regulator - and fuse it to the activation part for a different regulator (one which stimulates expression of the Gal1 gene). When you put this construct in the cell, you have the Gal1 promoter fusing to the Gal1 promoter for lacZ transcription. In the case of when no binding takes plate (ie. no lexA binding site).
Researchers demonstrate that the DNA binding site has its own ______________.
The catalytic site from a different TF can work as long as the binding site for the other TF is _____________________.
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