Last Class 1. Transcription 2. RNA Modification and Splicing
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Transcript of Last Class 1. Transcription 2. RNA Modification and Splicing
• 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 proteinsIs required before functional
Folding process starts at ribosome
Protein Folding PathwayMolecular Chaperone
An example of molecular chaperone functionsHsp70, 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 lymphocyteDifferent morphology, same genome
Six Steps at which eucaryotic gene expression are controlled
Double helix Structure
Regulation at DNA levels
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 interfaceIn general, many of them
will form between a protein and a DNA
DNA-Protein Interaction
1. Different protein motifs binding to DNA: Helix-turn-Helix motif; the homeodomain; leucine zipper; helix-loop-helix; zinc finger
2. Dimerization approach3. Biotechnology to identify protein and DNA
sequence interacting each other.
Helix-turn-HelixC-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 MotifsUtilizing 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
sequenceZinc 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 DimerSame 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 assayCan identify the sizes of
proteins associated with the desired DNA fragment
DNA affinity ChromatographyAfter 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 ImmunoprecipitationIn 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 RegulationTranscription
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 OperonCAP: 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 regions2. Transcription factors3. Chromatin structure
Gene Activation at a distance
Regulation of an eucaryotic geneTFs 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 recruitChromatin modulation proteins to induce transcription
Two mechanisms of histone
acetylation in gene regulationa. Histone acetylation
further attract activator proteins
b. Histone acetylation directly attract TFs
Synergistic RegulationTranscription 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