Regulation of Gene Expression

both lactose and glucose are present?

*no need to metabolize lactose* -some allolactose made -some lac represors do not bind to operator -BUT, glucose DOES supress adenylyl cyclase activity -so CAP does not bind to CAP site -RNA polymerase will NOT bind well to the promoter, so the operon is off -Catabolite Repression

mRNA translation

*phosphorylation* of the eukaryotic initiation factor, *eIF-2*, occurs when cellular *stress* occurs. This *inactivates eIF-2*, so that translation of proteins does not occur during these adverse conditions.*

mobile DNA elements

- DNA can move around the genome as *transposons* - movement mediated by transposase - DNA can be cut out directly, or it can be converted to an RNA intermediate and then back to DNA (*retrotransposon*). - This can increase the size of the genome - Most transposons can no longer move - Those that can could cause disease by landing in or near a gene and altering its expression (either positively or negatively) . - Examples are a certain type of hemophilia A, Duchene muscular dystrophy, and neurofibromatosis.

REGULATION OF EUKARYOTIC GENE EXPRESSION

- Much more complex than in prokaryotes; primarily transcriptional - Trans-acting molecules bind to cis-acting elements - also, gene expression is controlled by alternative mRNA splicing, mRNA stability, translational efficiency, and by protein stability, processing, and targeting. - Trans-acting molecules - transcription factors - proteins with DNA binding domains to *bind to the cis-acting DNA sequences AND an activation domain that binds to other coactivator proteins* - ^Combinatorial control - Eukaryotes only -a trans acting molecule can have a "repression domain" if it inhibits gene expression by recruiting co-repressors, so can have either stimulation or repression of transcription depending upon what the transcription factor recruits

RNA interference (RNAi)

- decreased expression of mRNA by increased degradation or decreased translation - Caused by micro RNAs (miRNA) - Precursor RNA (trigger) is cleaved by Dicer to miRNA - Guide strand of miRNA binds RNA-induced silencing complex (RISC) - miRNA (~ 22 nucleotides) base pairs with complementary sequence in mRNA target - bring RISC there - RISC can inhibit translation, or Slicer/Argonaute/Ago component of RISC can cleave mRNA - Making it inactive in translating protein. Has therapeutic potential. *Goal is to inhibit the proetin to be made by this mRNA either by cleaving the transcript or by inhibiting translation by the use of RISC*

Transcriptional regulation by cell surface receptors

- this explains how, e.g., peptide hormones can regulate the transcription of genes - glucagon binds to a cell surface; stimulates AC - increase in cAMP levels activates PKA - PKA phosphorylates CREB. - phosphorylated CREB binds to CRE - transcription of glucagon/ cAMP- responsive gene (such as PEP carboxykinase and others involeved in gluconeogenesis)

Stringent response

-amino acid starvation -want to slow down protein synthesis -Uncharged tRNA gets into A site of 70S ribosome -Triggers synthesis of *ppGpp by enzyme by activating Stringent Factor **(RelA)** that is part of ribosome* -This inhibits rRNA synthesis so that energy is not wasted and protein synthesis is slowed down when amino acids are at low levels. -inhibits transcription of rRNA, rRNA, mRNA

regulatory ribosomal proteins

-ensures balanced synthesis of rRNA and ribosomal proteins (r-proteins) -r-proteins bind with higher affinity to rRNA than mRNA -When rRNA present, ribosomes formed -*If rRNA levels drop, the r-proteins bind to the Shine-Dalgarno sequences of the polycistronic mRNA that codes for numerous rRNA proteins and inhibit their translation* -This prevents r-proteins from being made if they can't be used to make ribosomes.

Transferrin mRNA stability

-transferrin transports iron to cells that need it, transferrin receptor (TfR) bind transferrin to bring iron into cell when iron is LOW. LOW iron --> bring iron into cells Iron Response Element Binding Protein (IRP) -binds to Iron Response Element (IRE) on TfR mRNA 3' end making it more stable for translation. So iron can be brought into the cell. HIGH iron --> IRP binds Iron so this mechanism is shut down, TfR mRNA is not stabilized and it is degraded.

Eukaryotic Regulation by Co- and Posttrscriptional processing of mRNA

1) alternative splicing - exons can be included or skipped to make different proteins with different functions 2) mRNA editing - a base is altered after mRNA is completely processed (ex. ApoB100 vs. ApoB48)

CAP binding site

CAP binding site - where the catabolite gene activator protein (CAP; aka, cyclic AMP regulatory protein, CRP) *binds only when bound to cAMP* CAP enhances the ativity of RNA polymerase

Derepression

Derepression - the operator is bound by a repressor, that blocks the transcription of the gene by RNA Polymerase. *When gene expression is required, an inducer binds to the repressor, causes a conformational change, resulting in the repressor dissociating from the operator*. RNA Polymerase can now transcribe the gene. When the need for gene transcription ends, the inducer level drops and the repressor binds to the operator again, shutting down gene transcription.

Ferritin mRNA stability

Ferritin stores Iron, needed when iron levels are high, but not when low Ferritin mRNA has an IRE in its 5' end. LOW iron --> IRP binds to IRE, repressing translation cause it's not needed HIGH Iron --> IRP binds Iron, allowing translation of ferritin mRNA to proceed, therefore, ferritin levels increase and iron is stored until it is needed by the cells.

homeodomain

IMPORTANT -homeodomain proteins contain a protein structure called a homeodomain that binds to specific DNA sequences. This protein often regulates a cluster of related genes that have dramatic effects on differentiation and development, such as embryo polarity and body segmentation.

Lac Repressor is coded for by the?

LacI gene, which is *consitutively active*

Methylation of DNA

Methylation of DNA to form 5-methylcytosine silences DNA. Hypomethylated DNA is found in the promoters of active chromatin.

Only Glucose is present

Only glucose present.. no need for lactose Repressor protein will be bound to the operator Adenylate cyclase will be inactive so low cAMP levels CAP can't bind to cAMP so can't bind to CAP binding site to enhance RNA polymerase activity

Operon

Operon - sequential arrangement of genes with related function that is transcribed into a single polycistronic mRNA and is controlled by a common cis-acting DNA element, the operator

Histone deacetylase (HDAC)

Reverses the action of HAT, making histones bind DNA more tightly, condenses the chromatin to heterochromatin.

Cis-acting elements in Eukaryotic Transcription?

Use of common cis-acting elements near the promoter of coordinately regulated genes ensures simultaneous regulation of these related genes Remember: eukaryotes don't have operons

Cis-acting factors

are DNA sequences found (often) in the flanking, *non-coding regions* near genes that they regulate *on the same piece of DNA* Cis-acting elements are DNA sequences that are bound by trans-acting regulatory molecules. Cis-acting factors recruit the trans-acting factors that induce or repress transcription

Trans acting factors

are regulatory molecules (usually proteins) that are synthesized elsewhere and diffuse to the gene sequence that it regulates Transacting Molecules, usually proteins, are *synthesized from genes that are different from the genes targeted for regulation* Trans-acting molecules *bind to cis acting* elements on DNA **can be coded for by sequences that are on a completely different chromosome** than the cis-acting factor which it interacts

LacZ

codes for beta-galactosidase (cleaves lactose to glucose and galactose) lactose operon

LacY

codes for permease (to allow lactose to enter enter the cell) lactose operon

LacA

codes for thioglactosidase transacetylase (fxn unknown) lactose operon

Nuclear (intracellular) receptors

examples of trans-acting factors that bind to common elements to regulate a set of genes (e.g., glucocorticoid receptor, GR, binds to a glucocorticoid response element, GRE). 1) ligand-activated transcription factor 2) recruits coactivators to stimulate transcription 3) the GRE is an enhancer

Only lactose is present (no glucose)

need to get glucose from lactose metabolism Small amount of lactose --> allolactose (binds to repressor causing it to dissociate from DNA) This allows RNA polymerase to bind to the promoter and transcribe genes *Absence glucose, adenylyl cyclase is ACTIVE* AC activity *increase cAMP* CAP binds cAMP allowing bind to CAP binding site (aids RNA polymerase)

Histone acetyl transferase (HAT)

neutralizes positive charge on histone lysines, so they don't form salt bonds with the DNA phosphate backbone. makes DNA more accessibel to transcriptional machinery.

Lactose Operon

of importance when lactose is available for energy, but glucose (the preferred source for energy) is not. Three genes involved in lactose metabolism are sequentially arranged: lacZ, lacY, lacA

LacI

regulatory region, codes for the repressor protein (a trans acting factor) that binds to the operator site constitutively expressed and active (common Boards Q) LacI gene is not under the same regulation as the lac operon; LacI has its own promoter sequence that is not under regulatory control

Tryptophan (Trp) operon

repressor acts oppositely. Trp is an amino acid that the bacterium must synthesize. 1) When trp is present at high levels (no need to make it), it binds to the Trp repressor and shuts down five genes needed for its synthesis (negative control). When its level drops, it dissociates from the Trp repressor, which dissociates for the operator and allows the transcription of the genes needed for its synthesis. 2) **attenuation (Fig. 32.6) - sometimes RNA Polymerase initiates even when Trp levels are high. A hairpin forms at the 5' end of the mRNA kicking off RNA Polymerase.** **When Trp levels are low, ribosome stalls at two adjacent Trp codons, preventing hairpin for forming**

Promoter (P)

where RNA polymerase binds to initiate gene transcription

Operator (O)

where the lac repressor (LacI) binds The operator is downstream from the promoter, so that blocking the operator prevents RNA polymerase from initiating gene transcription