MB FINAL

Eukaryotic RNA Polymerases:

Have little or no intrinsic affinity for their promoters Almost always requires multiple activators (transcription factors, TF) proteins Each TF binds a short sequence A unique combination of a number of TFs is used to activate a given gene: General TFs and RNA Pol II bind the promoter region Transcription activators assist TF and Pol II to bind their promoter Activator-binding sites can be 1000s of bp away on the 5'or 3' side of the gene To achieve specific transcriptional activation of a gene, eukaryotes employ multiple regulatory proteins, or transcription factors, each of which binds a short sequence.

Some properties of riboswitch-regulated mRNAs

(1) Have unusually long sequences upstream from the translation start site that are necessary to form the riboswitch. (2) The upstream sequence is well-conserved in the same mRNA in different bacterial species, and sometimes in archaea, plants, and fungi. (3) Do not have protein binding partners, consistent with their ability to function in directly regulating gene expression.

Dosage Compensation Balances Gene Expression from Sex Chromosomes via three mechanisms.

(a) In mammals, one X chromosome of the female (XX) is inactivated, forming a compact structure called a Barr body. (b) In Drosophila, the single X chromosome of the male (XY) is transcribed at twice the level of the X chromosomes in the female (XX). (c) In C. elegans, the two X chromosomes of the hermaphrodite (XX) are transcribed at half the level of the single X chromosome of the male (X0).

DNA looping can be mediated by a single transcription factor

(a) the bacterial Lac repressor protein, a tetramer of identical subunits, binds two distant sites on a single DNA molecule, forming a DNA loop

Transcription corepressors

(b) Corepressors bind transcription activators and inactivate their polymerase-activating function. Note: The activator sites can also be located downstream of promoters

The promoter regions of higher organisms contain more control elements than those of unicellular eukaryotes, due to a need for:

- Changes in gene expression during development - Intercellular communication - Tissue specific expression

TFs are classified based on the presence of specific conserved motifs:

- 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

Combinatorial Control of Gene Expression

-the numbers of transcription factors as genome size and complexity increase -for example, yeast use roughly 300 transcription factors, C. elegans and D melanogaste rmore than 1,000 and humans more than 3,000 -although the number of TF increased with the number of genes there are still far fewer factors than there are genes to be regulated -combinational control allows higher eukaryotes to achieve exquisite specificity in gene regulation

Utiliity of Riboswitches and the stringent response.

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regulon

A group of genes or operons that are coordinately regulated even though some, or all, may be spatially distant in the chromosome or genome. e.g., regulation of a bacterial regulon: a) via production of an activator b) via destruction of a repressor.

Insulators / Boundary element:

A short sequence of DNA that prevents inappropriate cross-signaling between regulatory elements for different genes. -partition prevents cross-talk among regulators of promoters

Lac A:

A thiogalactoside transacetylase protein encoded by lacA gene. It modifies toxic galactosides that are imported along with lactose, facilitating their removal from cells.

Repression of eukaryotic (Pol II) gene expression.

A variety of repressor proteins can interfere with communication between Pol II and the DNA-binding transactivators, resulting in repression of transcription. Some proteins can act as an activator or coactivator at one promoter and a repressor or corepressor at another promoter. • Some repressor proteins interfere with communication between Pol II and the DNA-binding transactivators, resulting in repression of transcription. In fact, some proteins act as an activator or coactivator at one promoter and a repressor or corepressor at another.

The zinc finger motif (Type I):

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. The Zn2+ stabilizes the motif, which presents a recognition helix to DNA The Zn2+ does not directly interact with the DNA. Many DNA-binding proteins have multiple zinc fingers, which substantially enhance binding affinity by interacting simultaneously with the DNA

Summary of the stringent response:

Amino acid starvation leads to the binding of uncharged tRNAs to ribosome The stringent factor (RelA protein) binds the ribosome. RelA then catalyzes formation of the unusual nucleotide guanosine tetraphosphate (ppGpp). The rise in ppGpp concentration results in reduced rRNA synthesis The ppGpp binds RNA polymerase, blocking transcription of the rRNA genes. Need a bit more detail. Please read text.

Eukaryotic promoters use more regulators than bacterial ones

Bacterial promoters: - are typically near, or overlap, the promoter. - are usually regulated by only one or two transcription factors Eukaryotic genes, especially those of multicellular organisms, • Usually have numerous regulator-binding sites • Regulator-binding sites: - can span a large region (more than 50 kbp) - can be upstream, and/or downstream from the promoter, or even within the coding sequence of the gene itself.

Mechanisms of activation of eukaryotic (Pol II) gene expression.

Binding of Pol II to its promoter usually requires three types of regulatory proteins: 1. General (basal) TFs, which are required at every Pol II promoter 2. DNA-binding transcription activators also called DNA-binding transactivators, which bind to enhancers or UASs to facilitate transcription 3. Coactivators; these act indirectly by binding other proteins rather than DNA. The best-characterized coactivator is TFIID, which includes TBP and 10 or more TBP- associated factors (TAFs). Another important coactivator is the Mediator complex, which: o Is consist of 20 HIGHLY CONSERVED core polypeptides. o Is required for both basal and regulated transcription by Pol II o Stimulates phosphorylation of the Pol II general transcription factor TFIIH, enhancing Pol II efficiency.

Question: What is the relationship between glucose and cAMP? Why cAMP is low when glucose is high?

Glucose inhibits the synthesis of cAMP, and stimulates efflux of cAMP out of the cell. So, high glucose levels result in low cAMP levels, and visa versa.

LacZ

Lactose is converted to galactose and glucose, and a small amount of allolactose, the inducer of lac operon, by β-Galactosidase (encoded by lacZ)

For transcription to begin the preinitiation complex (PIC) must first be formed, which involves.

a.) binding of TATA-binding protien (TBP) to the TATA box -TBP is part of the larger TF complex called TFIID b.) TBP, then recruits additional general TFs and Pol II to form the PIC notes: -the minimal PIC is often insufficient for transcription initiation -positive regulation by TFs and coactivators is required to initiate the process -Pol II machinery is not uniform across all cell types. specific activators might be required in a specific tissue

Transcriptional regulation can be positive too

activator facilitates transcription example: an activator protein binds near the promoter and recruits RNA polymerase to initiate transcription

Coactivators

are required for communication between the DNA-binding transactivators and the complex composed of Pol II and the general TFs.

Transcriptional coactivators

coactivators and corepressors bins regulatory proteins without making direct contact with DNA. They act indirectly coactivators bind transcription activayots and facilitate their function in activating RNA polymerase

Transcriptional regulation can be negative

repressor inhibits transcription example: a repressor-binding site overlaps the promoter. When the repressor protein binds, RNA polymerase cannot initiate transcription and no mRNA is produced

Negative regulation of the lac operon by the Lac repressor.

when lactose is not present: -lac repressor binds the operator region -this prevents RNA Pol from binding the promoter site and transcribe the operon note: expression from lacI is constitutive when lactose is present, its metaboliye allolactose binds the lac repressor, inducing conformational changes that causes the repressor to dissociate from the operator RNA polymerase can then initiate transcription

Chromatin

• About 10% of the eukaryotic genome is in the form of heterochromatin: very condensed nucleosomes tightly packed together transcriptionally inactive often associated with structures such as centromeres and telomeres. The remaining, the less condensed is in the form of euchromatin nucleosomes spaced farther apart DNA can be decondensed and accessible to the transcription machinery Some of this DNA is transcriptionally active Fig 21-1. The two chromatin states are regulated by histone modifications (represented by red asterisks) and the binding of other factors (see Chapter 10).

The Lac operon encodes for three proteins that mediate lactose metabolism in E. coli.

LacY: LacZ: Lac A:

Example: The Pol II core promoter.

To initiate transcription RNA Pol II: • Requires the TATA box and the initiator sequence (Inr) • May also require the TFIIB recognition element (BRE), and the downstream promoter element (DPE).

Type II nuclear receptors

Type II nuclear receptors are located in the nucleus bound to DNA regardless of signal availability. The complex is inactivated by a coreprssor . Transcription is activated when the steroid hormone enters the nucleus and binds the complex at the HRE site. A coactivator replaces the corepressor, RNA Polymerase is recruited to the promoter site and transcription is activated. Example: the thyroid hormone receptor (TR) forms a heterodimer with the protein RXR to bind the HRE. The compelex is inactive without thyroid hormone, and is activated when the hormone is present.

The regulation of gene expression usually operates as....

a feedback circuit, and involves effectors.

certain transcription factors play an architectural role

some transcription factors, known as architectural regulators, bend the DNA when they bind their DNA site, thus promoting looping the refulator facilitates looping for recruitment of RNA polymerase by an upstream activator

The lactose (lac) operon of E. coli.

the lac operon includes three genes all three genes are transcribed as a single unit from the same promotor the operator region regulates transcription through interaction with a repressor protien, encoded by lac i the repressor: -has a separate promotor (is transcribed seperately) -is constitutively expressed `

Action at a distance: DNA looping

the regulatory site is far away from the promoter a transcription activator (blue) binds the regulatory site, a promoter/ RNA pol complex forming a DNA loop Transcription is activated

Example: Mechanism of TPP riboswitch function.

• When bound to TPP, the riboswitch assumes a conformation that sequesters the Shine-Dalgarno sequence, preventing translation • Mg2+ is required for TPP binding. It neutralize the negative charges on the pyrophosphate group of the ligand, and the RNA backbone, allowing closer packing of the RNA helices and TPP. • In the absence of TPP, the Shine- Dalgarno sequence is free and translation is initiated. • This is an example of induced fit.

Control of translation:

• mRNA must be processed and transported to the cytoplasm before translation; • Much more prominent role in eukaryotes than in bacteria • Why control expression at translation? - Repressed mRNA can be quickly translated

Graded control of the trp operon through transcription attenuation.

(a) Structure of the mRNA from the trp operon • The mRNA generated from the trp operon includes a 5' leader sequence containing four regulatory regions labeled 1 - 4. • "Sequence 1" is translated into the leader peptide, which regulates the trp operon. • The leader peptide has 14 aa, two of which are Trp. • When both free, sequences 2 and 3 can base pair to make a stem-loop structure. • 3 and 4 can base-pair to form a G≡C-rich stem-and-loop (hairpin) structure which 20 acts as a transcriptional terminator. (b) When [tryptophan] is high, [tRNA ] is also high. Thus ribosomes translate quickly through Trp codons of sequence 1 and into sequence 2. This allows sequences 3 and 4 to form a hairpin that stalls the RNA polymerase and terminates transcription. (c) In the absence of tryptophan: • Trp tRNA is low, and ribosomes stall on Trp codons of sequence 1, allowing sequences 2 and 3 to associate to make a hairpin. • With sequence 3 unavailable to associate with sequence 4, the terminator structure is not formed and transcription can proceed. • The amount of free tryptophan available for protein synthesis thus determines at what level the trp operon is transcribed. Note: The expression of the trp genes can be modulated ~70 fold by the repressor system alone, and up to ~700 fold by the repressor- attenuation system combined.

Review of Last lecture: Examples of transcriptional control in bacteria.

- Lac operon - Ara operon - Trp operon

Gene regulation can be regulated at seven points:

1.) transcription initiation 2.) RNA processing 3.) RNA stability 4.) protien synthesis 5.) protien modification 6.) protien transport 7.) protien degredation

LacY:

A membrane protein (Galactoside permease, encoded by lacY) allows entry of lactose into the cell.

An inverted repeat at the site of transcription factor binding.

A nucleotide sequence followed by the reverse, complementary sequence is known as an inverted repeat.

Type II zinc finger motif

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 eukaryotes, and there are a few examples among bacterial regulators. The Gal4 protein (Gal4p), a yeast transcription activator, is a dimer, held together by a leucine zipper.

Regulation of the ara operon.

CRP interacts with an activator/repressor to control transcription. Positive and negative regulation by the same molecule, the AraC protein. (a) When arabinose is absent and glucose is present (i.e. cAMP is low): AraC protein forms a dimer in which one monomer binds to araO2 and the other to araI1, forming a DNA loop. • This prevents RNA polymerase from binding and transcription from the ara operon (b) When arabinose is present and glucose is not (in which case cAMP is high) •AraC binds arabinose, and then activates the ara operon. •The AraC dimer changes conformation such that one monomer binds araI1 and the other binds araI2. Binding of AraC to araI2 recruits RNA polymerase to the promoter and activates transcription of the ara operon.\ Note: Arabinose is an "effector" for AraC.

The Yeast One-hybrid Assay system

Can detect the interaction of a TF (prey) with a DNA sequence (bait) The TF of interest is fused to GAL4 Activation Domain The Bait DNA is placed upstream of a reporter gene in host yeast The gene fusion is expressed in the host yeast If the "bait" and "prey" interact, RNA Pol II is activated and the reporter gene is expressed The reporter gene can be a selectable or screenable marker

Bacteria promoters include proximal regulatory regions.

E. coli RNA Pol binds DNA within a 100 bp (-70 to +30) region. Regulatory sequences are 5′-TATAAT-3′ box at ~ −10, TTGACA box −35, and the upstream promoter (UP) element at − 40 to − 60 region (for highly expressed genes) Eukaryotic promoters include both proximal and distant regulatory regions.

Some Activators Assemble into Enhanceosomes

Enhanceosome: A nucleoprotein complex of cooperating activators, which integrates regulatory information from multiple signals and generates a single transcriptional outcome at the target promoter. They allow a high level of specificity in the activation of the target gene through the cooperative binding of several activators. Example: Expression of interferon beta (IFNβ). At the IFNβ promoter, multiple activators present their activation domains together to simultaneously interact with the cofactor protein complex CBP-p300 The assembled enhanceosome recruits transcriptional machinery to the promoter region to initiate gene expression.

Multiple Regulators Provide Combinatorial Control of gene expression in eukaryotes

Example: Consider the following three promoters / genes. • Each promoter requires five different regulatory proteins. Each gene uses different combinations of transcription factors Some factors are used for more than one gene. (ex 7 unique regulator proteins bind to 15 DNA sites to control expresson of 3 different genes)

Strategy 3: Various concentrations of a group of protein regulators

Example: Gene expression in D. melanogaster oocyte /embryo • The D. melanogaster oocyte is surrounded by cells called "nurse cells" • The nurse cells secrete mRNAs encoding various TFs into the egg at specific locations, i.e., a gradient of mRNA for the different TFs is established within the egg. • During early embryonic development 3,000 to 6,000 cells form throughout the embryo. • Each cell traps the specific mRNAs present at that particular position in the embryo. • Each new cell thus produces a unique complement of transcription factors that act in a combinatorial fashion to express different proteins in the embryo. Example: Regulation of the eve gene, which produces a protein called even-skipped • Even-skipped is a transcription activator that promotes further differentiation in cells in which it is expressed. • Even-skipped is expressed only in specific cells of the embryo, generating a pattern of seven stripes that are visualized using a fluorescent antibody to even-skipped. Expression of eve is controlled by the concentrations of four proteins. Two are activators (Bicoid and Hunchback) and two are repressors (Giant and Krüppel). (Figure 21-14a) Different gradients of the mRNAs for these activators and repressors, established by the nurse cells, result in unique ratios of the four regulatory proteins in nearly every cell of the embryo. (a): The striped pattern of eve expression (revealed by antibody detection) is made possible by combinatorial control. (b) The relative levels and positions along the length of the embryo of even-skipped (top) and four TFs that regulate its expression (bottom).

Mechanism of Imprinting: An example

Fig. 21-18 Imprinting of the mammalian IGF2 gene. • CTC-binding factor (CTCF) binds the insulator of IGF2 • This enables insulator function and turns off transcription from IGF2 • Methylation of insulator sequences prevents CTCF binding to DNA • IGF2 gene can now be activated by its enhancer

The homeodomain DNA-binding motif.

First identified as a conserved 60 aa sequence in transcription activators that regulate body pattern development in fruit flies. 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 helix 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.

Blue-white selection using pUC vectors

Following cloning, vectors that took in the DNA of interest must be selected. Sometimes the vector does not contain the "insert" due to: -Contamination with the undigested vector -Vector re-ligating to itself. The blue-white selection technology allows selecting for bacteria that harbor the vector WITH insert. Transformed bacteria are grown on medium containing IPTG and X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside). And amp. Bacteria harboring pUC vector containing the insert DNA remain white. Why? • Bacteria harboring the empty pUC vector (i.e., no insert DNA) turn blue. Why?

Transcription Factors (TF):

Gene activation and repression in both bacteria and eukaryotes require transcription factors (also called transcription regulators). TFs: 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 structural motifs 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.

Regulation via catabolite repression: A positive regulatory system.

Glucose is metabolized directly by glycolysis and is E. coli's preferred energy source. When glucose is available, a regulatory mechanism known as catabolite repression restricts expression of the genes required for metabolizing other sugars (e.g., lactose, arabinose, etc.) even when these sugars are also present. In the absence of glucose cell's increase the expression of genes that allow the use of alternative food sources.

Role of high-mobility group (HMG) proteins

HMG protiens are nonhistone protiens that: -play an important structural role in transcriptional activation -are abundant in chromatin and bind nonspecifically to DNA -Bend DNA, helping form loops between distant enhancer and promoter elements note: high mobility refers to the rapid electrophoretic mobility of HMG protiens in polyacrylamide gells

Sets of genes may be regulated together in both Pro- and eukaryotes

In bacteria often groups of genes "involved in a common pathway" are transcribed together • Transcription produces a polycistronic mRNA regulatory sequences: activator binding site promotor and repressorbinding site (operator) -genes transcribed as a unit

Genetic Imprinting

In most diploid cells, both homologous genes (i.e., both alleles of a gene) are expressed equally. Some higher eukaryotes, however, have mechanisms to completely shut down the expression of an allele derived from one parent, using a process called imprinting. Imprinting is: - Is an inheritance process independent of Mendelian inheritance. - Epigenetic in nature - Affected genes are expressed in a parent-of-origin-specific manner - Is observed in fungi, plants and animals

Positive regulation of the lac operon by CRP.

In the absence of lactose the repressor binds the operator, blocking RNA polymerase and preventing transcription of the lac genes This is regardless of glucose availability and, thus, binding of CRP-cAMP to the operon. when glucose is high, cAMP is low, lactos present The repressor dissociates from the operator. However, low cAMP levels prevent CRP-cAMP formation and DNA binding. Transcription from the lac operon is weak. when glucose is low cAMP is high and lactose is present The cAMP levels increase in response to low glucose CRP-cAMP to bind the reg. region RNA Pol robustly binds and transcription from the lac operon is much stronger

Eukaryotic promoters have many regulatory elements

Insulators sequences: Sequences that interact with insulator proteins to block the action of activators on the promoter of a given (gene B). When the regulatory sites for gene A are filled, the activators could act on the promoter of gene A, but the insulator sequence blocks their action on the promoter of gene B.

Yeast two-hybrid analysis

Is based on ability of Gal4 protein (Gal4p) to drive the expression of yeast GAL genes - Gal4p has two domains; a DNA binding and an RNA Pol activating domain - Both domains are active on their own, but MUST be in close proximity To perform a two hybrid analysis A reporter gene is fused to the GAL promoter The a DNA binding domain of Gal4p is fused to a protein (protein X) The RNA Pol activating domain of Gal4p is fused to a second protein (protein Y) If protein X and protein Y interact, the DNA binding and activation domains of Gal4p ar brough together and can activate expression from the GAL promoter

Chemical structures of some effectors of the lac operon.

Isopropyl β-D-1-thiogalactopyranoside (IPTG) can also bind the Lac repressor and cause its dissociation from the operator, inducing transcription of the lac operon. IPTG is not a substrate for β-galactosidase. It is an "inducer" of the operon. The β-galactoside 5-bromo-4-chloro-3-indolyl-β-D- galactopyranoside (X-Gal) serves as a substrate for β-galactosidase, producing a blue color when metabolized. IPTG and X-gal are extensively used in research labs.

The helix-turn-helix DNA-binding motif.

Many bacterial and eukaryotic regulatory proteins use the helix-turn-helix motif to interact with DNA. The helix-turn-helix motif consists of about 20 amino acid residues that form two short α helices connected by a β turn. This motif lacks intrinsic stability and is generally part of a somewhat larger DNA- binding domain. Only one of the α-helical segments serves as the recognition helix.

SUMMARY

Most eukaryotic genes are inactive in their ground state, and require multiple activator proteins to stimulate transcription. The eukaryotic RNA Pol require activator binding to promoter sequences to activate gene expression. The cell produces only the activator proteins necessary for transcription of the subset of genes needed at that time. Many Pol II promoters include the TATA box and Inr sequences, as well as other sequences (enhancers in higher eukaryotes, and UASs in yeast) located far from the promoter. Regulatory proteins brings these sequences near the promoter by DNA looping . Transcription is stimulated by interactions between RNA polymerase core subunits and transcription activators (transactivators) bound to enhancer sequences. Often, coactivator complexes such as TFIID or Mediator act as bridges between the core transcription machinery and transactivators. 18

General transcription factors and RNA polymerase II bind the promoter

Note: General transcription factors are regulatory proteins required at every Pol II promoter. Also called a basal transcription factor. Activator-binding sites (regulatory sequences) can be distant from the promoter and located either before or after the gene. Activators bind regulatory sequences in DNA directly, whereas coactivators bind activators instead of DNA. Activation of Pol II is mediated by coactivators binding to core subunits of the polymerase through DNA looping. Fig. 21-3: A typical eukaryotic promoter.

Regulation of gene expression protein phosphorylation and cAMP.

Note: In eukaryotes cAMP is used differntly. Instead of binding directly to a transcription factor, eukaryotes use cAMP as a second messenger to carry a message received from outside to proteins inside the cell. cAMP is produced when a signal molecule binds a transmembrane receptor and induces it to activate adenylyl cyclase. Cyclic AMP-dependent protein kinase A (PKA) is repressed in its normal state. PKA becomes active upon binding of cAMP to this subunit. Activated PKA subunits enter the nucleus and phosphorylate various target proteins, such as CREB, which then recruits RNA polymerase to DNA.

For transcription to begin:

Pol II holoenzyme must be recruited to the promoter to form a preinitiation complex (PIC) with the general transcription factors. Assembly of PIC begins with the binding of TATA-binding protein (TBP) to the TATA box. TBP then recruits additional general transcription factors and Pol II. Note: TBP is part of the larger transcription factor complex that includes TFIID The minimal PIC is often insufficient for the initiation of transcription, and ositive regulation by transcription activators and coactivators is required. Note: The basal Pol II machinery is not as uniform across all genes; the individual components can vary with cell type.

A few notes on the state of gene expression.

Prokaryotic genes are mostly constitutively express: • 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 Eukaryotic genes are constitutively repressed and require activation in order to be transcribed. Why would this be? Eukaryotic DNA is condensed in the nucleus as chromatin (Nucleosomes) and often is inaccessible to RNA Pol. The chromatin state can have: • Open structure: Nucleosomes are often acetylated, and accessible to RNA Pol • Closed structure: Nucleosomes are methylated, and inaccessible to RNA Pol • Thus eukaryotic activators and repressors can act through modification of nucleosomes rather than by stimulating or inhibiting RNA Pol binding.

Notes:

Protein kinase A has many different target proteins that can lead to the activation or repression of various sets of genes. e.g., CREB (cAMP- responsive element-binding protein). • CREB is a transcription activator that is inactive when unphosphorylated. • When phosphorylated by PKA, CREB is activated and binds its CRE (cAMP-response element) site in the DNA. • CREB then activates transcription through a coactivator, CBP (CREB- binding protein). • CBP is a coactivator for numerous genes, including genes encoding other transcription activators, in many organs.

Riboswitches:

RNA itself has regulatory elements/regions known as Riboswitches • Exist within the 5′ untranslated region (5′UTR) of mRNA • Riboswitches are found in bacteria, archaea, and eukaryotes. They are most common in bacteria. • Consist of a small molecule-binding element connected to a regulatory region • Small specific metabolites binds the molecule-binding element. • Binding of the small molecule (the ligand) triggers a conformational change in the regulatory region, changing the shape of the entire RNA molecule.

Riboswitches can be very specific to their ligands.

Result of agarose gel electrophoresis of ribonuclease-cleaved samples (Fig. 20-17c). The glmS RNA was incubated with its known effector (glucosamine 6-phosphate) or the other compounds shown in (b). The riboswitch specifically recognizes GlnN6P Fig. 20-17c: Degradation of glmS mRNA triggered by different ligands. 10

The basic helix-loop-helix motif (b-hlh)

Share a conserved region of about 50 aa residues that are important for both DNA binding and protein dimerization. The b-hlh region contains two sets of amphipathic α helices: One set does NOT contain basic residues and mediates dimer formation One set contains basic residues and mediates DNA binding. The recognition helices grip the binding sequence in DNA in much the same way as the basic leucine zipper.

Global regulation of groups of genes.

Some genes are regulated in a coordinated manner.

Role of Nuclear Receptors in Gene Expression (e.g., steroids)

Steroid hormones must interact with nuclear receptors to induce gene expression. There are two major types of steroid-binding nuclear receptors:

Here we discuss three strategies:

Strategy 1: Different combination of the same TFs regulates different genes.

Other modifications are also important:

The C-terminal tails of the core histones are modified: - acetylation and methylation of Lys and Arg residues - phosphorylation of Ser or Thr residues - ubiquitination or sumoylation Ubiquitin and Small Ubiquitin-like Modifier (SUMO) proteins can attach to proteins in cells to alter their function.

Interaction of the Lac repressor and the Lac operatorn.

The Lac repressor is a tetramer formed from two homodimers. Each homodimer can bind one operator sequence. The lac operon contains three operator sequences O1 to O3. Only O1 is adjacent to the lac operon promoter. To repress the operon, one dimer binds to the O1 and the other dimer binds simultaneously to one of O2 or O3. The simultaneous binding of the Lac repressor to O1 and to O2 or O3 results in a looped DNA structure, providing an effective steric block to transcription. Note. Even when the operon is repressed, a few molecules of LacZ and LacY proteins are made. These help initiate lactose metabolism when it is available.

Compare and contrast the mechanisms by which TTP and glmS riboswitches inhibit translation of their RNAs.

The TPP-binding riboswitch alters mRNA conformation when TPP is bound, making the Shine-Dalgarno sequence inaccessible to ribosome binding. The glucosamine 6-phosphate-binding riboswitch also changes the conformation of its mRNA, but in this case the change activates a ribozyme ribonuclease function that cleaves and inactivates the mRNA.

leucine zipper motif

The basic leucine zipper motif is an amphipathic α helix, with a series of hydrophobic aa residues concentrated on one side of the helix. It is found in many eukaryotic and a few bacterial regulators A striking feature of this α 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. the basic leucine zipper motif. Often used to mediate protein-protein interactions in eukaryotic transcription factors. Certain TFs use leucine zippers in combination with basic residues at one end of the α helices that make up the recognition helices, forming basic leucine zipper motifs (AKA bZIP proteins) The aa sequences of several bZIP proteins. Notice the Leu (L) residues at every seventh position in the zipper region and basic residues (Lys (K) and Arg (R)) in the DNA-binding region.

Interaction of effectors with activators

The binding of effectors to activators can (a) inhibit or (b) enhance activation. (a) Activator 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.

Regulation via catabolite repression: A positive regulatory system pt2

The effect of glucose on expression of the lac genes is mediated by two elements: CyclicAMP(cAMP),asmall-moleculeeffector The activator cAMP receptor protein, or CRP, a homodimer which can bind both DNA and cAMP simultaneously. When glucose is absent, CRP-cAMP binds to a site near the Lac promoter and stimulates RNA transcription fiftyfold. CRP-cAMP is thus a positive regulatory factor responsive to glucose levels. This is unlike the Lac repressor, which is a negative regulatory factor responsive to lactose. The positive and negative regulatory systems act in concert.

Mechanism of Combinatorial Control in the expression of eve

The eve gene has five different enhancers, each with a complex array of binding sites for transcription activators and repressors • Only one enhancer needs to be active for eve to be expressed in a given cell. • Each enhancer is activated by a different combination of TFs in a particular cell type. The same four TFs are used by the five different enhancers in different ways.

Coordinated Transcription of several genes: The SOS system.

The expression of a number of genes involved in repair of damaged DNA in bacteria is induced in a coordinated manner. This is known as the SOS response. • The SOS response requires two key regulatory proteins: • The RecA protein • LexA repressor protein In the default (normal; no damage) state of the E. coli cell, the LexA repressor prevents transcription of the SOS genes. • In response to DNA damage, LexA undergoes autocleavage, inactivating itself and allowing transcription of the SOS genes. How is the autocleavage activity of LexA induced? Notes: • Autocleavage of LexA repressor requires RecA protein. • DNA damage creates sites of single-stranded DNA, which are quickly bound by RecA protein. DNA-bound RecA becomes a coprotease for LexA, and their association facilitates the destruction of LexA and induction of the SOS response.

Parental genomic imprinting of the human IGF2 gene.

The mouse igf2 gene, coding for the insulin-like growth factor II (IGF-II) is parentally imprinted, only the gene copy derived from the father is expressed. To know whether IGF2, the human homologue, is also imprinted, we used an ApaI polymorphism at the 3' untranslated region in order to distinguish between mRNA derived from each copy of the gene in placentae from heterozygote human fetuses, studied after careful removal of the decidua. Six term and two pre-term placentae of heterozygotes were studied, and in each case the cDNA contained only one of the two alleles present in the genomic DNA. In three cases the mother was homozygous for the non-expressed allele, allowing assignment of paternal origin to the transcribed gene copy. We conclude that, as in the mouse, human IGF2 is parentally imprinted.`

regulation of an r-protein operon via translational feedback mechanism (summary)

When [r-protein] is low relative to [rRNA], the translational repressor L4 molecules are NOT available to bind the mRNA and translation proceeds. When [r-protein] is higher than [rRNA], the repressor blocks production of additional r-proteins by preventing translation initiation.

Utility of the lac operon components in molecular biology.

When cloning a DNA fragment in pUC19, the LacZ gene helps select recombinant vector containing the DNA insert. How? What is needed for this selection to work?

Gene inactivation by histone deacetylation

When transcription of a gene is no longer required, acetylation of nucleosomes in that vicinity is reduced by the activity of histone deacetylases (HDACs), resulting in condensation of the chromatin to reduce or inactivate gene transcription.

Regulation of the Trp Operon

When tryptophan is abundant, expression from the trp operon is not needed. • Tryptophan serves as the effector molecule for the Trp repressor. • Association of tryptophan and repressor causes the repressor to bind the operator, blocking transcription.

coactivator

a protein that stimulates transcription by binding both the RNA polymerase and an activator or activators, without binding the DNA directly

Effector

a small molecule that binds a transcription activator or repressor, causing a conformational change in the regulatory protein that results in an increase or decrease in transcription from the gene (a) repressor (red) binds DNA in the absence of the effector. The effector binds the repressor causing its dissociation from DNA to permit transcription. (b) The repressor (orange) binds DNA in the presence of the effector, shutting down transcription.

Regulatory mechanisms unique to eukaryotes

nsulators are relatively short sequences, sometimes fewer than 50 bp Block transcription from unintended gene All insulator sequences in higher eukaryotes require for their functioning CTC-binding factor (CTCF) (see Figure 21-16).`

Hypersensitive sites

parts of the chromosome that are particularly accessible and sensitive to nucleases (DNAse I). This is because these areas are not tightly packed, due to relative absence of binding proteins such as histones. These regions and have little or no nucleosomes. Many hypersensitive sites correspond to binding sites for known regulatory proteins. The absence of nucleosomes in these regions may allow the binding of these proteins. In regions that are very active in transcription (e.g., rRNA genes) nucleosomes are entirely absent. Transcriptionally active chromatin also tends to be deficient in histone H1, which binds the linker DNA between nucleosome particles

overview of regulatory point in of gene expression

• In general, the transcriptional ground state (the inherent activity of promoters and transcription machinery in vivo in the absence of regulatory mechanisms) in eukaryotes is in a repressed state due to: - Chromatin structure - Large size of genome - Greater efficiency of positive regulation - Need for selective expression of genes in specialized tissue Thus, genes must be activated before transcription can commence

Riboswitches can distinguish between chemically related molecules based on:

• Atomic charge • Stereochemistry • The presence or absence of particular functional groups. Example: Ligand specificity in the glmS riboswitch. Note: glmS gene encodes an enzyme that catalyzes the conversion of fructose 6- phosphate and glutamine to glucosamine 6-phosphate. • The glmS riboswitch is a ribozyme. • On binding of its small-molecule metabolite, glucosamine 6-phosphate, the glmS precursor mRNA (pre-mRNA) degrades itself.

Role of guanine nucleotide-binding protein (G protein) in gene expression

• G proteins function as molecular switches in eukaryotic cells • When bound to GTP they are active; when GTP is hydrolyzed to GDP, G Proteins become inactive. • The signal transduction mechanism involves G protein-coupled receptors (GPCRs) that span the plasma membrane. • GPCR binds the signal molecule on the outside of the cell • Binding activates a G protein on the cytoplasmic side of the membrane. • When activated, G proteins promote the phosphorylation of proteins that in turn activate gene transcription. • G proteins mediate signal transduction for numerous hormones and other ligands.

The synthesis of r-proteins is coordinated with the availability of rRNA: the stringent response.

• In E. coli the seven rRNA operons respond to cellular growth rate and to changes in nutrient (particularly amino acids) availability. • This regulation is coordinated with amino acid concentrations, and is known as the stringent response. • The response ensures survival by redirecting cellular resources from growth and division toward the biosynthesis of amino acids. Fig. 20-19: Synthesis of rRNA is regulated by amino acid (aa) concentrations. When aa supplies are low, uncharged tRNAs enter the ribosomal A site, & trigger the stringent response, repressing rRNA synthesis. Read the text for more detail.

Regulation via translational feedback: Example: The r-protein operons

• In bacteria, an increased cellular demand for protein synthesis is met by increasing the number of ribosomes, i.e. r-RNA and r-protein. Note: At high growth rates, ribosomes make up about 45% of the bacterial dry weight. • Bacteria coordinate the synthesis of the ribosomal components (r-proteins and rNAs). This regulation occurs largely at the level of synthesis of r-proteins. • The r-proteins are encoded by 52 genes, present in ~20 operons of 1 to 11 genes. • One r-protein in each operon functions as a translational repressor, which binds the mRNA transcribed from that operon and blocks its translation. Note: In r-protein operons translation of one gene depends on the translation of others. When the repressor binds the r- protein mRNA, translation of all genes is blocked. The translational repressor has a higher affinity for rRNA than the r-protein mRNA. When levels of [r-protein] is low relative to [rRNA], few repressor protein molecules are available to bind the mRNA and translation proceeds. When [r-protein] is higher than [rRNA], the repressor binds the mRNA generated from the operon and blocks production of r-proteins by preventing translation initiation. Recall: In r-protein operons translation of all genes is blocked when the repressor binds the r-protein mRNA.

Strategy 2: Regulation of Transcription via Combinatorial Mixtures of Heterodimers

• Most eukaryotic TFs bind to DNA as homodimers. • However, several types of structurally related eukaryotic transcription factors can form heterodimers • A hypothetical family of four different but structurally related proteins could form up to 10 different dimeric species Example: the mammalian AP-1 TFs (e.g., Fos, Jun, and ATF) • AP-1 transcription factors control cell proliferation, differentiation, and programmed cell death. Some members of the Fos and Jun protein family are encoded by proto-oncogenes. • The protein-dimerization and DNA-binding regions of AP-1 family members are of the basic leucine zipper type. • AP-1 dimers activate genes containing an AP-1-binding site. Fos and Jun can function as heterodimmers

there are several classes of Riboswitches (RS)

• RS's are most common in bacteria, where they regulate genes related to synthesis of vitamins, cofactors, and amino acids • RS's are named after the ligand they bind • Over 15 classes are known, classified based on ligand type and RS secondary structure.

Posttranscriptional control of control of gene expression enables:

• Rapid up- or down-regulation of protein synthesis in response to molecular signals. • Why rapid? The cell does not have to wait for mRNA levels to change through transcription.

Regulation of the Trp Operon; fine-tuning of gene expression level

• The trp operon encodes five genes that control synthesis of tryptophan •Trp mRNA has a short half life (~30 min). This allows the cell to respond rapidly to changing needs for tryptophan • The trp operon is regulated by a repressor system and attenuation. In the absence of tryptophan the Trp repressor cannot bind the operator, and transcription from the trp operon is initiated so that Trp can be produced. The Trp repressor is a homodimeric protein

Type I:

• These are initially located in the cytoplasm complexed with Hsp70. • They relocate to the nucleus upon binding the hormone and release of Hsp70. • Upon release of Hsp70 the NR dimerizes and exposes a nuclear import signal sequence. • The NR-hormone complex then enters the nucleus and binds to a hormone response element (HRE) to activate transcription.

Riboswitches exert their control at the level of:

• Transcription: Alter RNA Pol binding. • Translation (b): Prevent translation by affecting SD sequences availability. •A third group are ribozymes.