Download Section on Gene Expression

Schedule for 501 Gene Expression Section 2008
Section Director, Dr. Peter Zassenhaus, [email protected]
Meets in LRC105 from 9-10 AM or from 9-11 AM on dual lecture days
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Lecture Date
Lecturer
Lecture Title
1
Tuesday, October 7
Zassenhaus
2
3
4
5
Wednesday, October 8
Thursday, October 9
Friday, October 10 (9 AM)
Friday, October 10(10 AM)
Zassenhaus
Zassenhaus
Zassenhaus
Zassenhaus
Overview and Bacterial Gene
Expression
Bacterial Gene Expression
Bacterial Gene Expression
Eukaryotic Gene Regulation
Eukaryotic Gene Regulation
6
Monday, October 13
Dorsett
RNA Processing
7
Tuesday, October 14
Eissenberg
8
Wednesday, October 15
Eissenberg
9
Thursday, October 16
Eissenberg
10
Friday, October 17 (9 AM)
Zassenhaus
11
12
13
14
Friday, October 17 (10 AM)
Monday, October 20
Tuesday, October 21
Wednesday, October 22
Eliceiri
Chang
Chang
Skowyra
15
Thursday, October 23
Skowyra
16
Friday, October 24
Note: meet in Schwitalla 409
Skowyra
17
Monday, October 27
9 - NOON
1
Chromatin Structure and Transcription
I
Chromatin Structure and Transcription
II
Transcriptional Repression
Summary and Review of Eukaryotic
Gene Regulation
Micro RNAs and RNAi
Protein Synthesis I
Protein Synthesis II
Protein Folding and Quality Control
Targeted Proteolysis as a Main
Regulatory Mechanism of Gene
Expression I
Targeted Proteolysis as a Main
Regulatory Mechanism of Gene
Expression II
EXAM
Section on Gene Expression
Transcriptional Regulation
Tuesday, October 7 – Friday, October 10
Zassenhaus, Doisy Research Center, Room 663 or 603, Ext. 7-8896,
[email protected]
Part I: Bacterial Transcriptional Regulation
Readings: On reserve in the Library are copies of the book “Genes and Signals” by Mark
Ptashne and Alexander Gann. Before class starts, please read Chapter 1, pages 11-42.
This is a very readable, small page size book with lots of good figures, not heavy on
facts, but superbly rich on ideas and explanations. This one chapter explains ALL of
transcription, gene regulation, AND cell signaling in all life forms! Once you understand
the principles presented here, then all the rest is just filling in the details. Our goal in
class will be to learn those principles. Before Class on Thursday, finish reading Chapter
1, pages 42-52. During Part 1, we will focus on how the binding properties of
transcriptional regulators determine how regulation works. Therefore, it will be helpful if
you review protein-protein interactions from earlier in the course, particularly the
quantitative aspects of measuring protein binding – i.e., the math of binding equilibria.
Over the course of the first three lectures (Tuesday thru Thursday) we will cover:
1. Structure and function of the bacterial RNA polymerase
2. Regulated Recruitment of transcriptional regulators:
Protein-DNA Interactions
Detecting physiological signals
Cooperative binding of proteins to DNA
Turning genes on or off – activation versus repression
3. Bacteriophage Lambda
The genetic switch of lysogeny versus lytic growth
Establishing lysogeny
Making an efficient switch – the importance of cooperativity
The repressor as a gene activator
DNA binding and synergy
4. Polymerase activation: NtrC and conformational changes in pre-bound polymerase
5. Promoter activation
2
Part II: Eukaryotic transcriptional regulation (Friday, 2 lectures; summarized,
reviewed and extended upon on Friday, October 17)
Although the principles utilized by eukaryotes to regulate gene transcription are the same
as you will have learned from examining bacterial gene regulation, the details are both
different and fascinating in their own right. Eukaryotes are also a marvelous example of
how simple principles can be combined into complex regulatory schemes. We will focus
on yeast as a model system to learn about how eukaryotes regulate gene activity. Before
class on Friday, please read Chapter 2, pages 59-103. In these two lectures, we will focus
on:
1. The structure of the eukaryote RNA polymerase machinery
2. Gal4 as a model transcriptional regulator
3. The nature of the activation domain in a transcriptional regulator
4. Recruitment of the RNA polymerase by transcriptional regulators
5. The role of nucleosomal modifiers in gene regulation
6. Transcriptional repression
7. Cooperative and combinatorial control of gene activity
After the next three lectures on Chromatin structure and its role in gene expression and
regulation, we will review transcriptional regulation in the first hour on Friday, October
19.
3
RNA Processing
Monday, October 13
Dale Dorsett, PhD., Doisy Research Center room 423, Ext. 7-9218
[email protected]
Production of mature messenger RNA and the mRNA life cycle
Readings:
Zorio, DAR, Bently, DL. 2004. The link between RNA processing and transcription:
communication works both ways. Exp Cell Res 296: 91-97
Concepts:
mRNA capping
mRNA splicing
reiterative splicing of long introns
mRNA polyadenylation
The RNA Pol II C-terminal domain and coordination of RNA processing
mRNA transport
mRNA localization
mRNA degradation and turnover
Degradation of missense mutant mRNA
Mechanisms of RNAi-induced degradation
4
Chromatin Structure and Transcription
Tuesday October 14 – Thursday October 16
Joel Eissenberg, Doisy Research Center, Room 421, Ext. 7- 9236
[email protected]
In each lecture, I’ll present some background material related to the key questions and
present experimental results from two research papers illustrating experimental
approaches to answering these questions.
Chromatin structure and gene activation I
All, or nearly all of the DNA in a eukaryotic nucleus is packaged into nucleosomes.
What is a nucleosome? How could the DNA-nucleosome interaction affect the ability of
DNA to be used as a template for transcription? How does a transcription factor interact
with a nucleosomal template?
Readings:
Li, G., and J. Widom (2004) Nucleosomes facilitate their own invasion
Nature Structural Mol. Biol. 11: 763-760
Owen-Hughes, T., and J.L. Workman (1996) Remodeling the chromatin structure of a
nucleosome array by transcription factor-targeted trans-displacement of histones. EMBO
J. 15: 4702-4712
Chromatin structure and gene activation II
Histones are covalently modified in vivo. These modification are correlated with various
aspects of chromosome activity, including gene regulation. How is chromatin modified
to facilitate gene activation?
Readings:
Peterson, C.L. and M.-A. Laniel (2004) Histones and histone modifications
Curr. Biol 14: R546-551
Kuo, M.-H., J. Zhou, P. Jambeck, M. E. A. Churchill, and C.D. Allis (1998) Histone
acetyltransferase activity of yeast Gcn5p is required for the activation of target genes in
vivo. Genes Devel. 12: 627-639
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Chromatin structure and repression
Methylation of lysines at certain sites on certain histones is correlated with gene
silencing. How are these methylated residues specifically recognized? How might
histone methylation contribute to gene silencing?
Readings:
Jacobs, SA.A., and Sepideh Khorasanizadeh (2002) Structure of HP1 chromodomain
bound to a lysine 9-methylated histone H3 tail. Science 295: 2080-2083.
Nielsen, S.J., R. Schneider, U.-M. Bauer, A.J. Bannister, A. Morrison, D. O’Carroll, R.
Firestein, M. Cleary, T, Jenuwein, R.E. Herrera, and T. Kouzarides (2001) Rb targets
histone H3 methylation and HP1 to promoters. Nature 412: 561-565.
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Review of Transcriptional Regulation
Friday October 17 – 1st hour
Zassenhaus, Doisy Research Center, Room 663 or 603, Ext. 7-8896,
[email protected]
Important principles of transcriptional regulation:
(1) Weak binding
(2) Cooperativity
(3) Combinatorial control
(4) Localization of gene activators
(5) Targeting by site-specific gene regulators
(6) Gene activation by recruitment
7
Gene regulation by microRNAs and RNA interference
Friday October 17 – 2nd hour
George Eliceiri, Doisy Hall 5th floor, Ext. 7-7863
[email protected]
Reading:
Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell
116:281-297.
Concepts:
What is RNA interference?
What are microRNAs?
Biogenesis of microRNAs
Gene silencing by mRNA cleavage
Gene silencing by translational repression
Gene silencing by heterochromatin transcription inhibition
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Protein Synthesis
Monday October 20 and Tuesday October 21
Yie-Hwa Chang, Ph.D., Doisy Research Center room 515, Ext. 7-9263
[email protected]
Suggested readings:
1. Cell and Molecular Biology, Kleinsmith and Kish (2nd edition), Chapter 11
2. Berg, JM, Lorsch, JR (2001) Mechanism of ribosomal peptide bond formation.
Science 291: 203
3. Ibba, M., Soll, D. (1999) Quality control mechanism during translation. Science 286:
1893
Part I: Mechanism of protein synthesis
1. Ribosome structure
2. Protein Synthesis
2.1 mRNA
2.2 tRNA
2.3 The initiation of protein synthesis
2.4 Peptide bond formation
2.6 Translocation
2.7 Termination of protein synthesis
2.8 Polysomes
Part II: The regulation of protein synthesis
Suggested readings:
1. Cell and Molecular Biology, Kleinsmith and Kish (2nd edition), Chapter 11
2. Pestova, TV, et al (2001) Molecular mechanism of translation initiation in eukaryotes.
Proc. Nat. Acad. Sci. USA 98: 7029
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3. Gale, M., Tan, S.L., and Katze M.G. (2000) Translational control of viral gene
expression in eukaryotes. Miocrobiol. Mol. Biol. Rev. 64: 239
1. Translational repressors
2. Life span of mRNA
3. RNAi
4. Phosphorylation
5. Availability of tRNA
5. Rate of termination of translation
7. Initiation factors.
10
Protein Folding and Quality Control; Targeted Proteolysis as a
Main Regulatory Mechanism of Gene Expression
Wednesday October 22 – Friday October 24 (three lectures)
Dorota Skowyra, PhD., Doisy Research Center room 407, Ext. 7-9280
[email protected]
The economics of protein synthesis, folding, and degradation: the evolutionary
solutions to optimize the cost of gene expression (Thursday)
Reading:
(1) Yewdell, J.W. (2001): Not such a dismal science: the economics of protein
synthesis, folding, degradation and antigen processing. Trends in Cell Biology, 11(7),
294-297.
(2) Schubet, U., Anton, LO.C., Gibbs, J., Norbury, C.C., Yewdell, J.W., Bennink, J.R.
(2000): Rapid degradation of a large fraction of newly synthesized proteins by
proteasomes. Nature 404, 770-774.
Overview: Once synthesized on the ribosome, every polypeptide needs to fold into a
conformation that ensures its designed function, modification and/or interaction with
other proteins. Frequently, such conformation involves multiple independently folded
modules and is hard to be achieved by spontaneous folding of the polypeptide itself.
Indeed, in the cell, protein folding is assisted by molecular chaperones, which in
multiple rounds of ATP-dependent binding and release “massage” the protein into its
optimal shape. If this process fails, the misfolded polypeptides are recognized by cellular
quality control systems and eliminated by targeted proteolysis via the ubiquitinproteasome pathway. A proteolytic pathway that recognizes and destroys abnormal
proteins must be able to distinguish between completed proteins that have “wrong”
conformations and the many growing polypeptides on ribosomes that have yet not
achieved their normal folded conformation. That this is not a trivial issue is
demonstrated by the observation that in normal growth conditions approximately one
third of newly synthesized proteins are degraded within minutes of their synthesis (Ref
2). Is this the best evolutionary solution to the problem of optimizing the cost of a
successful expression of a given gene? We will discuss this issue in class. PLEASE,
READ THE SUGGESTED LITERATURE AND PREPARE FOR DISCUSSION.
Problem (prepare your opinion to discuss in class): In your opinion, is the high rate of
degradation of the newly synthesized proteins the best solution to the problem of
optimizing the cost of a successful gene expression?
Ubiquitin-dependent proteolysis as a key regulator of the biological processes,
including gene expression (Friday)
11
Reading:
(1) Selected chapters from: Glickman, M., and Ciechanover, A. (2002): “The UbiquitinProteasome Proteolytic Pathway: Destruction for the Sake of Construction”, Physiol.
Rev. 82: 373-428. Chapter I: Introduction and overview, pp. 374-376. Chapter II: The
ubiquitin conjugation machinery, pp. 377-381. Chapter IV: Modes of substrate
recognition and regulation of the ubiquitin pathway, pp. 383-388.
(2) Conaway, R.C., Brower, C.S., Weliky-Conaway, J. (2004) “Emerging roles of
ubiquitin in transcription regulation”. Science 296, 1254-1258 (read for Friday).
Overview: Between the 1960s and 1980s protein degradation was a neglected area,
considered to be a non-specific dead-end process. Although it was known that proteins
do turn over, the large extent and specificity of this process, whereby distinct proteins
have half-lives that range from a few minutes to several days, was not appreciated. The
discovery of the lysosome by Christian de Duve did not significantly change this view,
because it became clear that this organelle is involved mostly in the degradation of extracellular proteins, and their proteases cannot be substrate specific. The discovery of the
complex cascade of the ubiquitin pathway revolutionized the field. It is clear now that
degradation of cellular proteins is a highly complex, temporally controlled , and tightly
regulated process that plays major roles in a variety of pathways during cell life and
death, including the regulation of gene expression, stress and immune responses, cell
cycle control and metabolic adaptation. We will discuss how the ubiquitin-mediated
proteolysis contributes to the regulation of gene expression.
Problem (prepare your opinion to discuss in class): how would you design an
evolutionary conserved system for intracellular protein degradation which would be
required to target 80% of total cellular proteins in a specific, regulated (when needed),
and timely (fast) manner?
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Exam
Monday October 27, 9 AM – Noon
A total of 80 points, 5 points per lecture
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