Transcript Document
• Last Class • • • • 1. Transcription 2. RNA Modification and Splicing 3. RNA transportation 4. Translation Quality control of translation in bacteria Rescue the incomplete mRNA process and add labels for proteases Folding of the proteins Is required before functional Folding process starts at ribosome Protein Folding Pathway Molecular Chaperone An example of molecular chaperone functions Hsp70, early binding to proteins after synthesis An example of molecular chaperone functions (chaperonin) Hsp60-like protein, late The Fate of Proteins after translation E1: ubiquitin activating enzyme; E2/3: ubiquitin ligase The production of proteins Summary • RNA translation (Protein synthesis), tRNA, ribosome, start codon, stop codon • Protein folding, molecular chaperones • Proteasomes, ubiquitin, ubiqutin ligase • Control of Gene Expression • 1. DNA-Protein Interaction • 2. Transcription Regulation • 3. Post-transcriptional Regulation Neuron and lymphocyte Different morphology, same genome Six Steps at which eucaryotic gene expression are controlled Regulation at DNA levels Double helix Structure The outer surface difference of base pairs without opening the double helix Hydrogen bond donor: blue Hydrogen bond acceptor: red Hydrogen bond: pink Methyl group: yellow DNA recognition code One typical contact of Protein and DNA interface In general, many of them will form between a protein and a DNA DNA-Protein Interaction 1. Different protein motifs binding to DNA: Helixturn-Helix motif; the homeodomain; leucine zipper; helix-loop-helix; zinc finger 2. Dimerization approach 3. Biotechnology to identify protein and DNA sequence interacting each other. Helix-turn-Helix C-terminal binds to major groove, N-terminal helps to position the complex, discovered in Bacteria Homeodomain Protein in Drosophila utilizing helix-turn-helix motif Zinc Finger Motifs Utilizing a zinc in the center An alpha helix and two beta sheet An Example protein (a mouse DNA regulatory protein) utilizing Zinc Finger Motif Three Zinc Finger Motifs forming the recognition site A dimer of the zinc finger domain of the glucocorticoid receptor (belonging to intracellular receptor family) bound to its specific DNA sequence Zinc atoms stabilizing DNA-binding Helix and dimerization interface Beta sheets can also recognize DNA sequence (bacterial met repressor binding to s-adenosyl methionine) Leucine Zipper Dimer Same motif mediating both DNA binding and Protein dimerization (yeast Gcn4 protein) Homodimers and heterodimers can recognize different patterns Helix-loop-Helix (HLH) Motif and its dimer Truncation of HLH tail (DNA binding domain) inhibits binding Six Zinc Finger motifs and their interaction with DNA Gel-mobility shift assay Can identify the sizes of proteins associated with the desired DNA fragment DNA affinity Chromatography After obtain the protein, run mass spec, identify aa sequence, check genome, find gene sequence Assay to determine the gene sequence recognized by a specific protein Chromatin Immunoprecipitation In vivo genes bound to a known protein Summary • Helix-turn-Helix, homeodomain, leucine zipper, helix-loop-helix, zinc-finger motif • Homodimer and heterodimer • Techniques to identify gene sequences bound to a known protein (DNA affinity chromatography) or proteins bound to known sequences (gel mobility shift) Gene Expression Regulation Transcription Tryptophan Gene Regulation (Negative control) Operon: genes adjacent to each other and are transcribed from a single promoter Different Mechanisms of Gene Regulation The binding site of Lambda Repressor determines its function Act as both activator and repressor Combinatory Regulation of Lac Operon CAP: catabolite activator protein; breakdown of lactose when glucose is low and lactose is present The difference of Regulatory system in eucaryotes and bacteria 1. Enhancers from far distance over promoter regions 2. Transcription factors 3. Chromatin structure Gene Activation at a distance Regulation of an eucaryotic gene TFs are similar, gene regulatory proteins could be very different for different gene regulations Functional Domain of gene activation protein 1. Activation domain and 2. DNA binding domain Gene Activation by the recruitment of RNA polymerase II holoenzyme Gene engineering revealed the function of gene activation protein Directly fuse the mediator protein to enhancer binding domain, omitting activator domain, similar enhancement is observed Gene regulatory proteins help the recruitment and assembly of transcription machinery (General model) Gene activator proteins recruit Chromatin modulation proteins to induce transcription Two mechanisms of histone acetylation in gene regulation a. Histone acetylation further attract activator proteins b. Histone acetylation directly attract TFs Synergistic Regulation Transcription synergy 5 major ways of gene repressor protein to be functional Protein Assembled to form commplex to Regulate Gene Expression Integration for Gene Regulation Regulation of Gene Activation Proteins Insulator Elements (boundary elements) help to coordinate the regulation Gene regulatory proteins can affect transcription process at different steps The order of process may be different for different genes Summary • Gene activation or repression proteins • DNA as a spacer and distant regulation • Chromatin modulation, TF assembly, polymerase recruitment • combinatory regulations