Mcb 2050 Midterm Topic 4

transcriptional, mRNA processing, translational, post-translational

presence of DNA motif in promoter, promoter accessibility, abundance of transcription factor, activity of TFs

the process of sensing external stimuli and conveying the information to intracellular targets to effect a response

trimerizes inresponse to proteotoxic stress, binds to heat shock elements (HSE) present as enhancers in promoters of heat shockprotein genes. Trimerization is repressed in non-stressed cells by intra-molecular interactions

lipid soluble steroid hormones pass through plasma membrane and bind intracellular hormone receptors, whichenter the nucleus and bind to hormone response elements (HRE) present as enhancers in hormone regulated genes. Fat soluble

extracellular peptide hormones bind to membrane bound receptors, which activates asignal transduction cascade (usually involving protein phosphorylation) that leads to TF activation. Water soluble

(TATA, GC, CAAT box) bind general (TBP) and basal transcription factors (NF1, Sp1) and and recruit RNA polymerase II. Enhancers bind activatingTF providing gene specific regulation of expression.

(i) act over large distances (ii) orientationindependent (iii) position independent (upstream, downstream of start site or within introns).

(i) function in short distances (ii) immediately upstream of initiation site. (iii) Position dependant, non functional if moved (iii) orientation dependant

modular proteins with domains required forDNA binding and transcriptional activation (some also contain dimerization or ligand binding domains).

for attachment to DNApromoter/enhancer elements

for binding other proteins requiredfor transcriptional activation

zinc finger motif, helix-turn-helix motif

leucine zipper, helix-loop-helix.

sequential or targeted deletions toidentify promoter sequences required for transcriptional regulation

radioactive DNAprobe containing binding site for TF mixed with cell extract and separated on non-denaturing gel. DNA probe bound to TFis shifted in mobility during separation. Technique will demonstrate the presence of a TF with the ability to bind the DNAprobe in the cell extract

(ChIP) demonstrate binding of TF (or other DNA-binding protein) to specific chromatin location in vivo. Cells treated with formaldehyde to cross-link proteins and DNA.DNA sheared and mixed with antibody (Ab) to specific TF. DNA/TF/Ab complexes purified, proteins removed andsequence of isolated DNA determined by sequencing or hybridization (ChiP-chip).

regulation of gene expression by changes in chromatin structure (heritable but reversible).

ensely packed transcriptionally inner, generally inactive

loosely packed transcriptionally active

occurs when a gene is transferred from euchromatic region to heterochromaticregion (or vice versa) resulting in altered gene expression. Drosophila mottled eye: inversion of X chromosome segmentmoving the white gene into a heterochromatic region in some eye facets but not others.

Thealteration of nucleosomes in preparation for transcription

HATs and SWI/SNF

acetylate histone lysine residues resulting inloosening of DNA-nucleosome interaction - more open chromatin facilitates TF binding and transcriptional activation.HDACs (histone deacetylases) remove acetyl groups resulting in chromatin compaction. Acetylation = more transcription

shifts nucleosomesalong DNA to clear promoter regions for TF binding.

attach methyl groups to cytosine residues inCpG dinucleotides to create C5-methylcytosine. Methylation of gene promoters (CpG islands) recruits chromatinmodifying proteins resulting in chromatin compaction and inhibition of gene expression

methylation pattern of a gene is determined by its parental origin. Igf2 gene is methylated in female germ line but notmale germ line. Only the copy inherited from the father is transcriptionally active.

dosage compensation of genes on X chromosome. mammals (XX/XY):in females one of the X chromosomes is inactivated. Drosophila (XX/XY): in males the X chromosome is hyperactivated.C. elgans (XX/XO): in hermaphrodites both of the X chromosomes are hypoactivated.

primary mRNA transcript can be processed to include or exclude different exon such that the various mature mRNAs can be translated into proteins with different functions

PABP1/eIF-4E mediated circularization prevents mRNA decay, deadenylation allows binding ofdeadenylating enzyme to destabilizing sequence AUUUA in the 3’UTR of short-lived mRNAs. Long lived have CCUCC sequence

Post-Transcriptional regulation

synthesized as pre-miRNA with ds hairpin structure, Drosha removes ends producing pre-miRNAthat is exported to cytoplasm, Dicer removes hairpin loop to produce short 22 nucleotide dsRNA, associates with AGO2protein to form the RNA-Induced Silencing Complex (RISC), strands are unwound, one is destroyed, the other (guide)base pairs with complementary sequence in specific mRNA 3’UTR resulting in silencing (translational inhibition or mRNAdegradation)

Made from same process

SiRNAs: maintain integrity of genome, defence against transposon,

MiRNAs: regulate gene expression

bind to exposed hydrophobic domains onunfolded proteins and prevent aggregation. Assist in protein folding and intercellular transport. Deliver irreversiblydamaged proteins to the ubiquitin-proteasome system (UPS). Regulate proteostasis

Damaged and short-lived proteins areubiquitinated and delivered to the proteasome for destruction.

HREs (Hormone response Element)

signal transduction

basal transcription factors, general transcription factors, RNA polymerase II

siRNAs (short interfering RNAs)

microRNAs

Position-effect variegation

HATs (Histone acetyl trangerases)

DNMTs (DNA methyl transferases), methyl, 5-methylctosine

Process = Gene imprinting

Base modification = methylation