Biology 366 Chapter 7

Why do cells need to regulate gene expression?

1. To respond to or adapt to changes in its environment 2. To generate different types of cells (differentiation) during development of multicellular organisms (refers to eukaryotic need)

How many E. coli genes are found in operons?

50% The remainder are solitary gene

What exactly is a CONSENSUS SEQUENCE?

A consensus sequence is one that represents the most frequent residues found at each position in a sequence element

How can transfection be used to test for the activity of the putative transcription factor?

A gene encoding a putative transcription factor is cloned into an expression plasmid. ***Remember that the transcription factor is the protein that binds to a control element that is located upstream from the gene that it affects. Plasmid 2 encodes a reporter gene (a gene that encodes for an expression that can be visualized--like beta-galactose)) with a minimal promoter (contains only one proximal control element --i.e. only has TATA box) and an upstream control element for the (****the upstream control element is the one that the transcription factor can recognize and binds to) Co-transfection of the two plasmids into cells that lack the proteins that encode the transcription factor... The expression of the reporter gene will increase if the transcription factor is an activator The expression of the reporter gene will decrease if the transcription factor is a repressor

Two-component regulatory systems in bacteria

A general two component regulatory system in bacteria consists of two domains that do different functions but work together to complete one function. The domains include a sensor domain and a response-regulator domain

Different types of cells express different genes.

All cells share certain genes that are specific for cellular survival and functioning, but cell types differ from one another in that they express different genes that make certain proteins that are highly specific for their particular function

Structure of pol II general transcription factors

All general transcription factors are multimeric proteins (several different subunits) designated TFIIA, TFIIB, etc One of the largest is called TFIID--it plays a crucial role and consists of 14 subunits. Of these subunits, one is called the "TATA-box binding subunit" and the 13 others are referred to as TBP-associated factors Another transcription factors is called TFIIH and functions with helicase activity which melts DNA at start site for transcription to begin.

RNA polymerase I, II, and III structure comparison

All three RNA polymerases have the 5 core subunits (beta, beta-prime, 2 alpha, and w) The same subunits that make up the bacterial RNA polymerase Only polymerase II (transcribes mRNAs) has the C-terminal domain (CTD) in its largest subunit

How does the Footprinting Assay work?

As stated before, the DNA fragments will contain the putative control element, such as fragments that include the start site and flanking sequences for a gene that has a TATA box control element By incubating the fragments with the TBP protein prior to the introduction of the endonuclease, the TBP protein creates a FOOTPRINT (PROTECTED REGION) SPANNING THE TATA BOX REGION. By incubating the fragments with the TFIID protein prior to the introduction of the endonuclease, the TFIID protein creates a footprint (protected region) at its associated control element sequence (which is located +20 base-pairs from the transcription start site) however, if INCUBATION OCCURS WITHOUT A PROTEIN, prior to DNase I digestion, the resulting DNA fragments are AN ARRAY OF BANDS REFLECTING RANDOM CUTTING (some sites are nonetheless more easily cut than others)

The control of eukaryotic gene transcription is much more complex

Aside from promoter elements that recruit RNA polymerase, eukaryotic genes have additional DNA elements that span a much larger region of DNA Transcription of a single gene is regulated by multiple DNA control elements and multiple transcription factors

Why does polymerase II transcription pause?

At most promoters, RNA polymerase II pauses after transcribing about 20 to 50 nucleotides The pause occurs because of the binding of a protein complex called NELF (negative elongation factor)

How is bacterial gene transcription commonly regulated?

Bacterial gene transcription is commonly regulated by SMALL MOLECULE LIGANDS

How do transcription factors actually activate RNA polymerase II?

Bridges are formed between the activator domains of the transcription factors and polymerase II via the MEDIATOR COMPLEX

Control of the transcription of the lac operon involves which three DNA regions

CAP site promoter operator **In order from upstream to downstream

And, the lac activator is:

CAP-cAMP, which binds to the CAP-site (which lies just upstream from the promoter) and interacts with the RNA polymerase to stimulate more transcription

What is the result of heterochromatin formation

Chromatin condensation blocks gene transcription at these sites

What causes heterochromatin formation?

Chromatin condensation is most often promoted by histone deacetylation and/or histone methylation **histone deacetylation removes the negative charges and promotes histone-to-histone interactions Protein factors required for heterochromatin formation were first discovered in yeast where one of two mating type genes were silences by heterochromatin formation.

Chromatin-Remodeling Factors

Chromatin-Remodeling Factors/complexes are required for many processes involving DNA (replication, recombination, repair, etc) Chromatin Remodeling factors are useful for the sliding of the nucleosomes in sequences that would generally bind to transcription factors. The chromatin remodeling factors promote the de-condensed nucleosomes to slide and they accelerate the binding of transcription factors to this area!

Eukaryotic Transcription is controlled by which two levels

Chromatin-level: closed or open? Promoter-level: DNA-binding transcription factors and other protein complexes assembles near gene promoters

Column chromatography techniques for purifying proteins

Column chromatography is commonly used to separate proteins from cell extracts into different fractions based on CHARGE, SIZE, OR HYDROPHOBICITY

The most important mechanism in determining whether a particular protein or RNA is produced:

Control of transcription INITIATION

Core promoter elements

Core promoter elements are recognized by the RNA pol II. They fall into 3 main categories: TATA Box Initiator Sequences CpG islands

Why is correct spacing between the two promoter elements important?

Correct spacing between the two promoter elements is important for recognition and binding by RNA polymerase One RNA polymerase molecule physically covers the entire region from -50 to +20 ...but the SIGMA SUBUNIT MAKES SEQUENCE-SPECIFIC CONTACTS ONLY WITH THE -35 AND -10 ELEMENTS

How do DNA-Binding Proteins regulate gene transcription?

DNA-Binding Proteins BIND TO THE PROMOTER and other control regions of the genome and act either as REPRESSORS or ACTIVATORS of gene transcription

How many sigma factors does E. coli have?

E. coli has a total of 7 sigma factors--each recognizing a specific consensus sequence (some bacteria have as many as 63 sigma factors)

Electrophoretic Mobility Shift Assay (EMSA)

EMSA is a type of column chromatography. After the cell extracts are ran through the column and separated into fractions, THE COLUMN FRAGMENTS ARE INCUBATED WITH A RADIO-LABELED DNA FRAGMENT CONTAINING A CONTROL ELEMENT (probe). The labeling is then followed by GEL ELECTROPHORESIS. When a transcription factor binds to the control element, (it obviously becomes heavier!) so a slower migrating (not as far down) protein-DNA complex is observed **EMSA is useful for detecting transcription factors during purification

Each promoter element consists of what kind of sequences?

Each E. coli promoter element consists of a specific CONSENSUS SEQUENCE of about 6 to 10 base pairs long

Regulation by elongation factors

Elongation factors, such as NELF (pauses transcription), DSIF (procession of elongation), provides regulation of elongation The regulation of elongation provides an additional mechanism for controlling gene transcription in addition to transcription initiation

Roles of the enhancer sequences

Enhancer sequences STIMULATE TRANSCRIPTION OF ANY GENE, even when located thousands of base-pairs away from the start site of that gene. **The location can vary and the enhancer sequence can still be effective. The enhancer sequence can be upstream, downstream within introns, or even after the last exon. **Enhancers are OFTEN RESPONSIBLE FOR TISSUE-SPECIFIC TRANSCRIPTION OF GENE

Enhancer sequences

Enhancer sequences are distant control elements They often stimulate transcription

Characteristics of enhancer sequences

Enhancer sequences are generally 50 to 200 base-pairs long and consist of multiple individual control elements Each of the individual control elements within the enhancer sequence is about 6 to 10 base pairs long. The individual control elements are positioned close to one another. **Thus, a single enhancer sequence binds multiple transcription factors.

ER

Estrogen response element

Footprinting Assay--the technique::

First you incubate a radio-labeled DNA fragment. The DNA fragment must contain the putative control element. Only the 5' end of the fragment is tagged You then add small amounts of DNase I, producing about 1 random cut per DNA fragment. ****IF THE TRANSCRIPTION FACTOR, that can bind to a control element found within the DNA fragment sequences, IS PRESENT in the protein sample (the cell extract), THE BINDING SITE IS PROTECTED from cutting by the endonuclease. DNA fragments are then separated by gel electrophoresis (fragment size indicated the distance from the labeled end).

Specific control factors binding characteristics

For the most part, each single specific transcription factor binds to only one specific control element Remember that "control factor" = the protein that binds to the DNA And, "control element" = the sequences of the DNA that the control factor recognizes and binds to. One specific control element is of a specific sequence of 6 to 10 base pairs long.

Critical elements

Further analysis of control regions using single base-pair changes shows that CRITICAL ELEMENTS CONSIST OF SPECIFIC SEQUENCES OF ABOUT 6 TO 10 BASE PAIRS LONG. These critical element consensus sequences serve as a binding site for a specific transcription factor

What kind of genes generally require alternate sigma factors?

Genes that require alternate sigma factors generally control a specific functions, many times these functions relate or are caused by environmental stresses

How are genes within a single operon regulated?

Genes within a single operon are COORDINATELY REGULATED (one promoter region)

More transcription factors = more complex uniqueness

Given the abundance of transcription factors, as well as the multiple corresponding control elements for each gene, transcription of each gene can be regulated in an extremely unique manner.

What is a heterochromatin?

Heterochromatin reflects high-level chromatin condensation

There is an ABUNDANCE of specific transcription factors in eukaryotes!

Humans encodes over 2,000 specific transcription factors, while yeast encode for several hundred. Therefore, while the complexity of an organism is not correlated to the number of genes, the complexity may be correlated to the number of control factors---dictates the amount of ways that the genes can be expressed.

Different cell types and their different transcription factors

Importantly, different cell types accumulate a specific set of transcription factors that bind to specific control elements (a major factor in determining which genes are expressed and how the genes are expressed).

What is the mediator complex is absent?

In absence of a mediator, only minimal basal transcription can occur

"Promoters" of eukaryotic DNA

In eukaryotes, the term promoter generally refers to those DNA control elements that lie close to the start site and recruit as well as position RNA pol II for transcription initiation The region close to the start site is sometimes referred to as the "core" promoter

Examples of actions performed by small molecule ligands

In some cases, small molecules induce phosphorylation of repressors or activators that modulates their DNA-binding properties. **Note that some activators, like CAP, control transcription of several genes

In E. coli, the sigma factor...

Is responsible for recognizing and binding to the -10 and -35 promoter elements and must first associate with the RNA polymerase before it can bind to the promoter Transcription of most genes in exponentially growing (not starvation, etc.) E. coli cells requires the sigma-70 factors

What can transfection tell you about the transcription factor?

It can tell you whether or not the transcription factor of interest is a repressor or an activator.

How can chromatin condensation by transcription factors to regulate gene transcription?

It is associated with some repressor domains of some transcription factors Promoter-localized chromatin condensation: UME6 transcription factor binds to URS1 upstream control element The repressor domain of the UME6 transcription factor recruits a protein complex that has deacetylase activity localized nucleosome condensation occurs, which prevents polymerse II from binding to TATA box

Where can heterochromatin be found?

It is readily observed in telomere and centromere regions where there is no gene transcription Chromatin condensation also occurs at many other sites along the chromosome

Examples of lipid soluble hormones

Lipid soluble hormones include steroid hormones: cortisol estrogen testosterone glucocorticoid retinoid thyroid hormones

Transcription factors that bind as dimers:

Many bacterial repressors and any eukaryotic transcription factors bind as dimers! These bacterial repressors commonly contain a DNA-binding domain called a helix-turn-helix motif... and this is what causes it to bind as a dimer!

Gene expression is regulated at:

Many different levels: 1. Initiation (most important for determining whether a particular protein/RNA is even produced) 2. transcript processing 3. Transport 4. mRNA stability 5. translation

The promoter elements of E. coli:

Most E. coli genes contain 2 promoter elements (one positioned at -35 and the other at -10 bp upstream of the transcription start site)

What are nuclear receptors?

Nuclear receptors are transcription factors that are activated by the lipid-soluble hormones that can cross the cell membrane. Because these lipid-soluble proteins can simply pass through the cell membrane, these nuclear receptors are directly activated by the hormones themselves

RNA polymerase II is formed by a pre-initiation complex in vitro--the following is the ordered assembly

ONLY THE TBP SUBUNIT OF TFIID IS REQUIRED TO INITIATE ASSEMBLY in vitro (complete TFIID is required in vivo) this pre-initiation complex is assembled on the TATA box of promoters (that contain them) in vitro Once bound, TBP recruits TFIIB which then recruits the RNA polymerase II enzyme bound TFIIF TBP bends DNA and then the TFIIH has helicase activity that melts the DNA at the start site There is then a release of general transcription factors, except for TBP. Phosphorylation of RNA polymerase II CTD is required before elongation can start

Polymerase I and III have their own set of general transcription factors

Polymerase I and III transcription relies on specific sets of general transcription factors that recognize DNA control elements Since Polymerase I and III only transcribe a few gene, the control elements are much simpler Transcription rates by polymerase I and III are tightly associated with cell growth and proliferation... where as transcription rates by polymerase II is associated with the amount of transcription factors and the contacts made with the mediator complex!

General transcription factors

Polymerase II is actually recruited to the gene start sites using these general transcription factors General transcription factors are required at all promoters used by pol II--these are distinct from the "specific transcriptional factors" discussed above.

Promoter Elements

Promoter elements are short, specific sequences (generally 6 to 10 base pairs long) located in the promoter regions. The promoter elements are recognized and bound to by the regulator proteins

How can promoter-localized decondensation be used by transcription factors?

Promoter-localized de-condensation is used to activate genes For example: the GCN4 transcription factor binds to USA upstream control element The activation domain of the GCN4 transcription factor binds to its control element and then recruits a complex with acetylase activity. This facilitated the assembly of polymerase II at the TATA box

RNA pol II's CTD

RNA pol II is the only RNA polymerase that has a C-terminal domain (CTD) in its largest subunit Pol II CTD consists of 7 AMINO ACID REPEAT SEQUENCE Try-Ser-Pro-Thr-Ser-Pro-Ser This is repeated 26 times in yeast and 52 times in vertebrates Pol II CTD phosphorylation plays a crucial role in the INITIATION OF MRNA TRANSCRIPTION and is also REQUIRED FOR RECRUITING ENZYMES INVOLVED IN MRNA PROCESSING

Eukaryotic gene transcription involves 3 different types of RNA polymerases:

RNA polymerase I RNA polymerase II RNA polymerase III

RNA polymerase I

RNA polymerase I transcribes pre r-RNAs pre r-RNAs function to encode ribosome components and function in protein synthesis (translation)

RNA Polymerase II

RNA polymerase II transcribes mRNA, snRNA, siRNA, and miRNA mRNA--encodes proteins snRNA--RNA splicing siRNA--chromatin-mediate repression/translation control miRNA--translation control

Polymerase III

RNA polymerase III transcribes tRNAs, SSrRNA, snRNA U6, 75RNA, and other stable, short RNAs tRNAs--protein synthesis (transfer for translation) ss rRNA--ribosome component, protein synthesis snRNA U6--RNA splicing 75 RNA--signal-recognition particle for insertion of polypeptides in teh endoplasmic reticulum other stable short RNAs--various functions--unknown for many

Regulation at transcription elongation

Regulation of gene expression also occurs at the elongation stage of transcription. The transcription of a gene/operon can be reduced by preventing the elongation of the transcript--this is called attenuation

Repressor Proteins

Regulator proteins that "repress" transcription, by either preventing RNA polymerase from binding at the start site, or preventing it from transcribing

Activator Proteins

Regulator proteins that positively affect the transcription of a gene by either enhancing the recruitment of RNA polymerase to the start sites, or stimulating the activity of the already bound RNA Polymerase

The repressor domain of the transcription factor:

Remember that transcription factors are repressors, while others are activators. The transcription factors that repress transcription have repressor domains in place of the activator domains (as discussed in the above examples) These domains inhibit, rather than activate, gene transcription Like activator domains, repressor domains, work independently from the DNA-binding domain of the transcription factor. If mutations occur within the repressor domains of the genes that encode transcription factors, then this often lead to constitutive (unregulated) transcription of the genes they are designed to control!

The characteristics of response elements:

Response elements either contain indirect repeats or direct repeats and bind dimers of nuclear receptor proteins There are 5 types of response elements: *****GR, ER, VDR, TR, and RARE For GR and ER, the ligand binding occurs in the cytoplasm **This promotes translocation of the receptor to the nucleus For VDR, TR, and RARE, heterodimer nuclear receptors are found exclusively in the nuclues **these repress the gene they bind to in the absence of their hormone ligand

How do transcription factors bind to control elements?

Same principles of binding in both prokaryotes and eukaryotes Non-covalent interactions occur between the specific amino acids of the binding domain of the transcription factors and the specific sequences of DNA bases

Sequence-Specific DNA Affinity Chromatography

Sequence-Specific DNA Affinity Chromatography is often used as a last step in purification of transcription factors. **The DNA control element is actually covalently linked to the column material Partially purified transcription factor samples (they are partially purified because they've already gone through multiple steps of purification and the sequence specific is the last step as stated before) are added to the top of the column under physiological salt concentrations and allowed to flow through. The corresponding transcription factor binds to the attached DNA while the contaminating proteins (proteins that do not contain the specific sequence) continue to flow through the column The transcription factor, which is now trapped in the matrix of the column is recovered by raising the salt concentration of the matrix--the high salt level disrupts the binding.

What are Sigma Factors and why are they important

Sigma Factors are essential for the bind of RNA Polymerase to DNA. They are the "part of the RNA polymerase" that makes the actual contact with the DNA at specific sequences.

What is the role of small molecule ligands in bacterial transcription?

Small molecule ligands alter DNA binding properties of repressor and/or activator proteins (lac operon is one example) This kind of gene transcription regulation allows bacterial cells to respond to cellular needs (i.e. needs of different nutrients based on availability)

Regulation of the the transcription factor activity

So the transcription factor's purpose is to regulate transcription, but it's activity is also often regulated. The first part of activating a transcription factor often involves transmembrane receptors that sense extracellular responses This signal then triggers a reaction cascade leading to activation or repression of transcription factors already present in the cell (this often involves phosphorylation) In some cases, extracellular signals involve lipid-soluble molecules that diffuse through cellular membranes and directly activate transcription factors by binding to and changing the conformation of the activation domain

E. coli and DNA looping in transcription:

Some E. coli activator proteins bind far from the promoter and DNA looping is required

Specific Transcription factors

Specific transcription factors are ESSENTIAL for regulating eukaryotic gene transcription. **Only basal level transcription (very low level transcription) takes place in the absence of specific transcription factors **Specific transcription factors (as opposed to general transcription factors) are required for regulation of gene transcription levels

Where do specific transcription factors bind?

Specific transcription factors bind to specific DNA control elements further away from the start site where RNA POLYMERASE II PRE-INITIATION ARE ASSEMBLED

So how do the nuclear receptors then bind to the DNA?

The DNA has specific binding sites (control elements) for nuclear receptors that are referred to as RESPONSE ELEMENTS

Describe how the DNA-Binding Proteins specifically Binds to the DNA

The DNA-Binding proteins (regulator proteins) recognize and bind to SHORT, SPECIFIC DNA SEQUENCES IN PROMOTERS and other control regions (these DNA sequences are referred to as promoter elements)

The transcription-factor's DNA-binding domain:

The DNA-binding domain determines which genes can be stimulated for transcription There is only one DNA-binding domain per transcription factor protein Therefore, one transcription factor recognizes one specific sequence of a control element of a specific gene.

What are the types of DNA-binding domains of transcription factors

The DNA-binding domains of eukaryotic transcription factors can be grouped based on their related structure with similar motifs The three best known structural types of transcription factors are: *homeodomain *zinc finger *leucine zipper

PAX6 genes and anirida (lack of iris)

The Pax6 gene regulates a number of developmental pathways. Some mutations that cause one defective Pax6 gene (heterozygous) leads to anirida. Anirida is a congenital disease (a disease you are born with) that causes a lack of irises in both eyes.

The transcription factor's activation domain:

The activation domain(s) work when attached to any transcription factor DNA-binding domain (as both of these domains are needed to complete a whole transcription factor). There may be more than one activation domain per transcription-factor protein. **activation domains function by interacting with the RNA polymerase II initiation complex via a mediator protein complex.

All Nuclear receptors share a common domain structure

The common domain structure contains two copies of a C4 zinc-finger motif... This domain works to bind the ligands (lipid soluble hormones) The domain functions as an activator or repressor in some cases

The drug Tamoxifen

The drug Tamoxifen is used to treat breast cancer under the concept of the estrogen-binding conformational changes. Basically, when estrogen is present it binds to the activation domain of the estrogen receptor, causing a conformational change. This conformational change allows for the activation of RNA polymerase II via a co-activator protein. Tamoxifen binds to this same estrogen binding site of the activation domain of the estrogen receptor, however instead of activating transcription, it block the required conformational change

Example of gene activator protein that requires DNA looping in E. coli:

The glutamine sythetase gene A (glnA) promoter is recognized by sigma-54 polymerase. The activator protein for the transcription of glnA is called NtrC. NtrC (activator) binds to so-called ENHANCER SEQUENCE The ENHANCER SEQUENCE lies far upstream of the promoter (at about -108, -140) Looping of the DNA allows NtrC to bind to the sigma-54-polyerase and ACTIVATE TRANSCRIPTION

Helix-turn-helix motif

The helix-turn-helix motif is the DNA-binding domain of the bacterial repressors. Specific contacts between amino acid side chains in the recognition alpha-helix (sequence-reading helix) and bases in the major groove of DNA determine the binding affinity between the transcription factor and its specific control element sequence

Homeodomain of a Transcription Factor:

The homeodomain transcription factor is a DNA-binding domain of a transcription factor that is a consensus sequence of about 60 amino acids long and has a similar structure and the bacterial repressor's helix-turn-helix motif ***homeodomains are most often found in transcription factors that function during development

Leucine Zipper motifs of a Transcription Factor

The leucine zipper transcription factor is a domain with an alpha helices and the hydrophobic Leu amino acid at every 7th position... This causes it to form coiled-coil dimers Not only found in the binding domain of a transcription factor, but also in many other proteins Basic amino acids at one end of the dimer bind to specific sequences in the adjacent major grooves like a pair of tweezers... or more like a grilling fork that binds to two control elements with a space in between Since alpha helices have about 3.6 residues per turn, the leu at every 7th position makes one side of each hydrophobic (the Leus are on the same side, every other turn), this facilitates the coiling that occurs around each other to form a dimer (like a zipper)

What does the magnitude of transcription of a given gene reflect?

The magnitude of transcription of a give gene reflects the combined effects of all the contacts--the contact between the transcription factors and the control elements, and the contact between the transcription factors bound to the promoter and the enhancers with the mediator complex. As well as, the contacts made with the TAF subunits of the TFIID protein with the mediator

What is the mediator complex, though?

The mediator complex is a multisubunit co-activator complex... that forms bridges between the activator domain of the transcription factor and polymerase II.. allowing for interaction!

Again what are the contacts that are made between the transcription factors and the mediator

The mediator complex makes contact with 1) the multiple transcription factors (that are bound to promoter-proximal elements and enhancers). These transcription factors make contact with one or more of the subunits of the mediator complex **requires DNA binding 2) the TAF subunits of TFIID protein

Tamoxifen is an antagonist

The normal ligand (estrogen in this case) is called the agonist, and the the inhibitor of the binding is called the antagonist

Structure of Eukaryotic RNA polymerases

The overall structure of bacterial and eukaryotic RNA polymerases is similar However, the eukaryotic polymerases have 12 to 16 subunits, in contrast to the bacterial RNA polymerase enzyme, which only has 5 core subunits

The promoter region of each bacterial gene (or operon) usually binds to how many regulator proteins?

The promoter region of each bacterial gene or operon usually binds to ONE OR TWO REGULATOR PROTEINS (activators or repressors)

What is the role of the promoter region?

The promoter region serves as a BINDING SITE for RNA POLYMERASE and REGULATORY PROTEINS

Where are promoter regions located, in reference to the gene?

The promoter regions lie upstream of the gene transcription starts sites

What is the promoter-recognizing subunit of the RNA polymerase referred to as?

The promoter-recognizing subunit of the RNA polymerase is called the sigma factor. The core of the RNA Polymerase (2 alpha subunits, one Beta, and one Beta-prime) must associate with the promoter-recognizing subunit (sigma factor protein) before the RNA polymerase itself can bind to its specific DNA promoter elements. The binding of theta to the core RNA polymerase positions the RNA polymerase in the correct start site

Yeast, a less complex eukaryotic organism, also has multiple transcription-control factors (both a proximal and a distant control element) but the variability and complexity in yeast transcription regulation, compared to higher eukaryotes, is much simpler

The proximal control element in yeast is usually the TATA box. However, the TATA box in yeast is further upstream as compared to the higher eukaryotes (about 90 base-pairs, instead of about 25 to 35 base-pairs upstream) The distant control element in yeast is the upstream activating sequence--This is what we call the enhancer sequences in mammals. However, in mammals the enhancer sequence consists of multiple control elements, whereas the yeast usually contain ONLY ONE DNA CONTROL ELEMENT and the UAS is it!

Response Protein

The response protein is phosphorylated and activated by an activated sensor-domain. This creates a conformational change **in the case of the histidine kinase sensor, the phosphate on the His residue is quickly transferred to a specific Asp residue in the response regulator. There is an effector domain on the response regulator protein. This effector domain binds to specific promoter to activate transcription.

Sensor Protein

The sensor domain does not bind to the promoter, but if it senses the presence of a nutrient (or a nutrient binds to the sensor protein) it will activate a pathway (i.e. the histidine kinase pathway is activated). The activation of this pathway phosphorylates and activates the response protein.

Regulation of Gene Transcription in Bacteria is relatively simple!

The steps follow:

There are various mediator complexes.... They all share some of the same subunits, while also differing in others

The subunits that are the same in all mediator complexes are those that bind to polymerase II or that maintain the overall structure of the mediator complex. These subunits are necessary for all mediator complexes to function and they are always required for transcriptional control of all genes Then, there are some subunits that differ between mediator complexes... and these subunits are those that bind to specific activation or repressor domains of transcription factors and are thus required only for the activation of certain genes.

How are heterochromain formed in telomeres?

The telomere genes of yeast contain a specific telomere repeat sequence that bind multiple copies of the RAP1 protein The Rap 1 protein recruits a complex Sir Proteins, one of which displays deacetylase activity Removal of histone acetyl groups (which contain negative charges) from neighboring nucleosomes PROMOTES CHROMATIN CONDENSATION (less negative charges = less repel)

What determines the transcription level of each gene?

The transcription level for each gene is a result of the combined effects of multiple transcription factors bound to its control elements. Example:: The transcription level can be measured in a unit such as "mRNA copies synthesized per unit of time"... and the effects of the multiple transcription factors can enhance transcription (allow for more mRNA copies to be synthesized in less time) or slow it down, etc.

What do promoter regions govern?

The transcription level of the gene and its response to regulatory signals

How is the transcription of the lac operon is prevented when lactose is absent?

The transcription of the lac operon is prevented by THE BINDING OF THE LAC REPRESSOR PROTEIN (specifically the tetramer) TO THE OPERATOR REGION

Example of expression regulation at elongation

The tryp operon When tryptophan is available as a nutrient, a tryp repressor protein reduces transcription of the operon (as in the lac operon) Further reduction of tryp operon is achieved by prevent elongation of the transcript

VDRE, TRE, raRE

These are heterodimer response elements that are found only in the nucleus The function to repress the genes they bind to in the absence of their associated hormone ligand

Initiator Sequences

These promoter elements lie closer to the start site than TATA boxes--in fact this class of promoters actually contains an initiator sequence that overlaps the start site Initiator sequences are poorly conserved compared to TATA boxes

GR and ER Response elements

These response elements bind to their associated hormone ligands in the cytoplasm This binding then promotes the translocation of the receptor to the nucleus

TR

Thyroid hormone response element

Mapping of tissue-specific transcription-control-regions (typically enhancer sequences)

Tissue specificity of expression in the developing embryo can be revealed by linking different segments of the mouse gene (Pax6) to a reporter gene (beta-galactose). Then you introduce this construct to fertilized mouse eggs to create a transgenic mouse The gene expression can be followed throughout development to understand the alternative transcription start sites or other control elements that are used to created different expressions of the gene--the same gene, under different control elements, can produce different types of tissue types.

More about the domains of transcription domains

Transcription factors always have only one DNA-binding domain (Which makes sense, because it should only need to recognize one specific sequence within a DNA fragment--otherwise the whole system would be over-complicated). Transcription factors, however, may contain more than one transcription-activation domain The order of these domains differs depending on the factor, but the domains do work separately--they do not depend on one another to do their function!

Modular nature of transcription factors: Modular: composed of standardized units or sections for easy construction or arrangement

Transcription factors are composed of individual units that work specifically for different functions of the transcription factors. Transcription factors have two functions, typically: 1 for transcription-activating, and the other for DNA-binding. Each of these functions reside in different domains of the protein Transcription factors' domains not only reside in separate domains, they also function separately.

Transcription Factors

Transcription factors are the repressors and activators of transcription that bind to DNA control elements in eukaryotes

Transgenic Mice

Transgenic mice are much simpler to produce than gene knockouts **How to make transgenic mice: *foreign DNA, that contains genes of interest, is simply injected into fertilized eggs *During development of the fertilized egg, multiple copies of the foreign DNA inserts randomly into the host chromosomes. (They insert via nonhomologous recombination--obviously non-homologous because they are not from same source and so sequences will most likely not be nearly identical.) *about 10 to 30 percent of the offspring will contain the foreign DNA in chromosomes of all their tissues and germ line. You then have to isolate those mice (the ones expressing the foreign DNA) to propagate the DNA in the germ line.

Uses of transgenic mice

Transgenic mice carrying the dominant negative alleles can often be used to inactivate specific gene products Transgenic animals and plants are also useful to make protein products for commercial purposes (GMO foods scare some people--and they should duh).

True or False: Promoter regions are required for gene transcription.

True! No promoter= no transcription

How can you detect specific transcription factors in cell extracts?

Using a technique called Footprinting Assay, you can detect specific transcription factors in cell extracts.

VDR

Vitamin D3 response element

Lac Operon transcription levels when lactose is present and glucose is present/absent?

When glucose and lactose are present, transcription of the lac operon is low, because some lac operon repressor proteins remain bound. When lactose is present, but glucose is absent, the lac operon transcription is high because CELLS RESPOND TO THE ABSENCE OF GLUCOSE BY SYNTHESIZING cAMP.

How does cyclic-AMP (cAMP) affect the transcription of the lac operon?

When glucose is absent, the cells are more dependent on lactose as their carbon source. So, in order to make the lac operon transcription more efficient, cells begin to synthesize cyclic AMP. cAMP binjds to the catabolite activator protein (CAP) cAMP-CAP complex binds to the CAP-site (which is located just upstream of the lac promoter) and interacts with the RNA polymerase to stimulate more transcription

When lactose is absent, what steps are taken to control transcription?

When lactose is absent there is no need to waste the energy to transcribe the lac operon (and ultimately make the enzymes used to break it down and metabolize lactose). Thus, transcription of the lac operon is prevented.

When lactose is present, what steps are taken to control transcription?

When lactose is present, lactose actually BINDS TO THE LAC REPRESSOR PROTEIN. This CAUSES A CONFORMATIONAL CHANGE OF THE REPRESSOR PROTEIN and its DISSOCIATION FROM THE OPERATOR.

Zinc-Finger motifs of a Transcription Factor

Zinc-Finger motifs are common in transcription factors (often in multiple copies) but are also found in other cellular proteins Cysteines and histidine (C2H2 or C4) in the motifs bind zinc ions via their side groups A C3H2 zinc finger is a 23-26 amino acid long consensus sequence A C4 zinc finger is a 55-56 amino acid long consensus sequence

CpG Islands

about 60% to 70% of genes are not transcribed as frequently as the ones containing the TATA box or an initiator sequence (many of the housekeeping genes that are required for basic metabolic processes) These genes utilize a CpG island promoter sequence The CpG island is a GC-rich region and is about 100 to 1000 base-pairs long Transcription in CpG island initiate at several "alternative sites" within this GC-rich region and transcribes in both directions Transcription in the "wrong" direction (antisense) is frequent, but often does not proceed as far as transcripts; this divergent transcription may play a role in transcriptional regulation

The sigma protein factors is not actually a part of the RNA polymerase core

but it does have to bind to it for transcription to occur

TATA Box

commonly found in highly transcribed genes located 25 to 35 basepairs upstream of the start site contains an 8-nucleotide-long consensus sequence

What types of DNA control elements exist in eukaryotes?

each eukaryotic gene has 2 distinct sets of of DNA control elements One set is promoter proximal and the other set is distant These distinct sets of DNA control elements that can bind to specific transcription factors when these are present in a given cell type When bound to a control element, the specific transcription factors either stimulate or repress transcription of the genes that possess this control element by interacting with RNA pol II either directly or indirectly

Lac Operon

encodes 3 proteins that metabolize lactose LacZ encodes B-galactose--the first gene in the operon

DNA-Binding proteins regulate what?

gene transcription

GR

glucocorticoid response element

So, the lac repressor is:

lac repressor protein, which binds to the lac promoter to inhibit transcription

The structural features of Transcription Factors' activation/repressor domains:

many activation domains are rich in acidic amino acids (asp, glu); others are often rich in glutamine or proline Some activation domains, especially acidic ones, remain unstructured (do not fold into a specific tertiary structure) until they bind to a co activator protein. Some activation domains are highly structured but require binding to a small ligand to change to an active conformation.

linker-scanning mutation analysis

maps the promoter-proximal control elements it identifies transcription control elements in the promoter region

Why map promoter-proximal control elements?

promoter regions from any gene can be linked to a reporter gene for analysis (a fluorescent protein gene) the reporter gene allows for expression of the gene, and how that expression is affected by the control elements (control region) to be visualized.

Summary of transcription in bacteria

repressor and activators bind to specific sequence elements in promoter regions binding of repressors and activators is often modulated by binding of small molecules two-component systems rely on sensor proteins to activate repressors or activators some operons are also regulated by attenuation (transcript terminated prematurely)

Alternate sigma factors

required by a small number of genes in E. coli that have promoter elements that bind to sigma factors other than sigma-70 Alternate sigmas are often used for gene activation in response to environmental stress!

RARE

retinoic acid response element

The size of bacterial promoter regions are

small--Rarely contain more than 2 or 3 promoter elements

Attenuation

transcript terminated prematurely reduction of the transcription of an operon or gene/ achieved by preventing the elongation of the transcript this control is mediated by formation of alternative base-paired structures within the first 140 nucleotides of the transcript, one of which leads to termination

Transcription resumes:

transcription resumes and elongation proceeds following binding of DSIF and further phosphorylation of the Pol II CTD domain, as well as phosphorylation of NELF and DSIF

Bacterial promoter regions are located:

upstream of gene starts