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