October Blood Drive ~ VIP/American Red CrossSlide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Important themes in the function of DNA-binding proteinsSlide 16Slide 17Slide 18Slide 19Slide 20Slide 21Slide 22Slide 23Slide 24Slide 25Slide 26Slide 27October Blood Drive ~VIP/American Red CrossFriday (10/24) Room 104 // Illini Union12pm– 4pmenter drawing to win one ofTEN GIFTCARDS to BUFFALO WILD WINGScontact:[email protected] of gene regulationGene regulation refers to the molecular mechanisms that control whether or not a gene is expressed as RNA and/or protein.•Some genes are only expressed when the cell encounters certain environmental signals.•In multicellular eukaryotes (e.g. animals; plants), some genes are only expressed by one or a few cell types.•Some genes are only expressed during specific phases of the life cycle, e.g. during early development of the embryo.InductionRepression1 2 3 nCondition- +Stimulus- +StimulusLevel of expressionGenes that produce a relatively constant level of RNA and/or protein under all conditions undergo constitu-tive expression.[Typical of “housekeeping genes” that are essential for cell function.]Genes whose expression is actively regulated undergo facultative expression.EXAMPLEE. coli does not synthesize the enzymes needed to metabolize lactose unless there is lactose available in its environment. If lactose is added to the growth medium of starving cells, there is a thousand-fold increase in their expression of the gene that encodes the -galactosidase enzyme.Some pancreatic cells express the peptide hormone insulin (red). Others express the peptide hormone glucagon (green). Cells outside the dotted line express neither peptide.Tissue section of a human pancrease. The nuclei of individual cells are stained blue.The gene Sonic hedgehog (Shh) is primarily expressed in embryos.In this chicken embryo, the Shh mRNA is stained black by in situ hybridization. This gene is transiently expressed in small clusters of skin cells that will develop into feathers as the animal matures.How do cells regulate gene expression?The amount of protein produced by a gene can be regulated at a variety of different steps:DNA pre-mRNA mRNA ProteinTRANSCRIPTION RNA TRANSLATION PROCESSING RNA PROTEINDEGRADATION DEGRADATIONThe most common means of gene regulation is to control the rate of transcription, i.e. the number of transcripts a gene produces during a given interval of time.The most common means of gene regulation is to control the rate of transcription, i.e. the number of transcripts a gene produces during a given interval of time.* *Advantages of regulating gene expression at transcription:•The cell does not waste resources in the synthesis of unneeded RNAs or proteins.•Cells generally have only 1 (haploid) or 2 (diploid) copies of a gene. Thus, regulating transcription requires relatively few regulatory molecules.Disadvantage of regulating gene expression at transcription:•When a transcriptionally silent gene is activated, it can take a long time for protein to accumulate.DNA is an information storage molecule. It has effectively no enzymatic activity of its own. The chemical reactions that comprise transcrip-tion are dependent on proteins which bind to and interact with the DNA.In bacteria, the RNA polymerase holoenzyme can act by itself to recognize promoters and initiate transcription. This is known as basal tran-scription.But for many genes, regu-latory proteins can bind to DNA in the vicinity of the promoter and either decrease (= repress) or increase (= activate) the rate at which polymerase initiates transcription.Watson, Fig. 18-6activatorAn important note on nomenclature ...Watson's textbook defines 'promoter' as "the DNA sequence that initially binds the RNA polymerase" (pg. 432). However, in practical terms most scientists use the term 'promoter' to refer to the entire regulatory region of the gene, i.e. that portion of the DNA which binds proteins that influence the gene's transcription. To circumvent this ambiguity, the site bound by RNA polymerase can be distinguished as the core promoter.DNA-binding proteins that regulate the rate at which RNA polymerase initiates transcription are referred to in general as transcription factors. This term is used more widely in eukaryotes, but in MCB 250 we will use it for both prokaryotes and eukaryotes.Important themes in the function of DNA-binding proteins•Specificity - Transcription factors (TFs) display sequence-specific binding: i.e. they only bind to DNA that has a specific nucleotide sequence.•Non-covalent bonds - When it binds to DNA, the TF does so by forming multiple non-covalent bonds. •Chemical affinity - The cumulative strength of these non-covalent bonds determines the ‘chemical affinity’ between the TF protein and its DNA binding site.adapted from Watson, Fig. 6-13aThe cI transcription factor has six different DNA-binding sites within the w phage genomeConsensus sequenceNote: none of these six binding sites perfectly matches the consensus sequence. However, they are close enough to consensus that they can all bind the cI protein.#1#2#3#4#5#65'- T A T C A C C G x C G G T G A T A - 3'3'- A T A G T G G C x G C C A C T A T - 5'Watson, Fig. 7-33The binding of protein to DNA can be studied experimentally by means of the electrophoretic mobility-shift assay (EMSA).In EMSA a short piece of labeled dsDNA (the ‘probe’) is run on a polyacrylimide gel in the presence (right lane) or absence (left lane) of a potential DNA-binding protein.If this protein securely binds to a nucleotide sequence within the DNA probe, their combined molecular weight produces a second, more slowly migrating band on the gel.If there is no protein or the protein doesn’t bind this DNA sequence, the labeled DNA will migrate on the gel as a single band of uniform size.probe probe +The placement of protein