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

Different morphology, same genome Neuron and lymphocyte Different morphology, same genome

Six Steps at which eucaryotic gene expression are controlled

Regulation at DNA levels Double helix Structure

Hydrogen bond donor: blue Hydrogen bond acceptor: red 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 Different protein motifs binding to DNA: Helix-turn-Helix motif; the homeodomain; leucine zipper; helix-loop-helix; zinc finger Dimerization approach 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

Utilizing a zinc in the center An alpha helix and two beta sheet 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

Zinc atoms stabilizing DNA-binding Helix and dimerization interface 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)

Same motif mediating both DNA binding and Protein dimerization 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 Enhancers from far distance over promoter regions Transcription factors 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 Histone acetylation further attract activator proteins 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