Nucleic Acid Biology

BBSG 501
Section 2 -Nucleic Acid Biology
Fall 2003 Semester
Section Director: Ali Shilatifard, Ph.D.
Office: R105: ext. 8137: e-mail: shilatia@slu.edu
Lecturers:
H. Peter Zassenhaus, Ph.D.
Office: R418: ext. 8443: e-mail: zassenp@slu.edu
Joel Eissenberg, Ph.D.
Office: DH 121: ext. 8154: e-mail: eissenjc@slu.edu
Dale Dorsett, Ph.D.
Office: DH First floor: ext. 8124: e-mail: dorsettd@slu.edu
Yie-Hwa Chang, Ph.D.
Office: MS 130: ext. 8150: e-mail: changyh@slu.edu
Dorota Skowyra, Ph.D.
Office: DH 102: ext. 8140: e-mail: skowyrad@slu.edu
John Tavis, Ph.D.
Office: MS R421: ext. 8441: e-mail: tavisje@slu.edu

DATES: 9/15/03 – 9/16/03 Lecturer: Ali Shilati , Ph.D.
Reading: Maniatis, Chapter 14 (will be handed out in class), Molecular Biology of the
Cell Chapter 7. P 291-334.
Lecture 1: Overview of Molecular Techniques/Structure of the
Eukaryotic Nucleus
Monumental Steps in the Development of Recombinant DNA technology
1-Isolation of DNA
2-Discovary of DNA polymerase
3-Discovary of DNA re-naturation
4-Discovery and usage of restriction nucleases
5-Elucidation of genetic code
6-Discovary of reverse transcriptase
7-Cloning of cDNA
8-Discovery of DNA hybridization methods
9-DNA sequencing
10-Polymerase Chain Reaction (PCR)
11-EST libraries
12-DNA sequencing using CE
13-Development of MALDI methods for protein identification
14-Sequencing the genome human and other organisms
15-Your future contributions
DNA polymerases and their usage in molecular biology
A. I. Mammalian DNA polymerase
a) The discovery
b) The Mechanism of action
B. II. Taq DNA polymerase
a) Advantage of its use
b) Disadvantages of its use
C. III. Taq DNA polymerase with proof reading ability
Use of Taq DNA polymerase in PCR
a)Subcloning of cDNA and DNAs
b)DNA sequencing via Taq and PCR
c)Cloning the end of genes
d)Generation of mutation and deletion with DNA polymerase

Lecture 2: Structure of the Eukaryotic Nucleus
Intracellular Compartments and Protein Sorting
A. The Compartmentalization of Higher Cells
I. Intracellular compartments
II. Relative amounts of membrane
B. The Protein Movements Between Compartments
I. Vesicular transport
II Transmembrane transport
III Gated transport
C. Typical Signal Peptides
D. Experimental Approaches for Defining Signal Sequences
I. Protein fusion method
II. Signal exchange method
E. The Nucleus
I. Nuclear structure
II. The nuclear pore complex
F. Transport to Nucleus
I. Nuclear localization signal (NLS)
II. Diffusion through the nuclear pore
III. Nuclear import through glucocorticoid receptor
IV. Reaction mechanism
V. Type of polymerases (I, II, and III)
VI. Discovery of RNA polymerases

DATE: 9/17/03 Lecturer: Ali Shilatifard, Ph.D.
Lecture 3: Nucleic Acid Biology
Reading: Molecular Biology of the Cell pp. 421-434
Cellular RNA is Synthesized by RNA Polymerases
VII. Reaction mechanism
VIII. Type of polymerases (I, II, and III)
IX. Discovery of RNA polymerases
Basal Transcription Machinery and Their Purification
I. The promoter element and the TATA box
II. Basal transcription factors
III. Preinitiation complex formation
IV. Promoter clearance
V. Transcription elongation
VI. Transcription termination
Transcriptional Activation
Transcriptional Enhancers and Regulation of Gene Expression
I. The gene control region of typical eukaryotic gene
II. The modular structure of a gene activator protein
Transcriptional Repression
I. Competitive DNA binding
II. Masking the activation surface
III. Direct interaction with basal transcription machinery
DATE: 9/18/03 Lecturer: Ali Shilati, Ph.D.
Lecture 4: Nucleic Acid Biology
Reading: Review by Shilatifard
Regulation of Transcription Elongation
Transcription Initiation and Promoter Clearance
Factors involved in the initiation of transcription
Factors involved in the promoter clearance

DATE: 9/18/03 Lecturer: Ali Shilati, Ph.D.
Lecture 4: Nucleic Acid Biology (continued)
RNA polymerase II General Elongation Factors
Classes of elongation factors
Assays of transcription elongation
Purification of the Pol II elongation factors
RNA Polymerase II Elongation and Human Cancer
Elongation and VHL
ELL and Leukemia
DATES: 9/19/03 & 9/22/03 Lecturer: H. P. Zassenhaus, Ph.D.
Lecturers 5 & 6 Nucleic Acid Biology
Required readings from your textbook are: Molecular Biology of the Cell, 3rd edition;
Mechanism of Transcription: pp 223-227 and pp 365-371
Regulation of Transcription pp 401-408; 417-420; 443-444
Post-transcriptional regulation: pp 453-468
Please read your text before coming to class.
Lectures on Bacterial Gene Regulation and the Mechanisms of mRNA
Stability in Eukaryotes
We will also discuss issues presented in two review articles which will be handed out
ahead of class. These should be read after Lecture 1 but before Lecture 2!
1) The Functional and Regulatory Roles of Sigma Factors in Transcription, Gross et al.,
Cold Spring Harbor Symp on Quant Biology, Vol. 63, 141-155 (1998)
2) mRNA Stability in Eukaryotes, Mitchell and Tollervey, Current Opinions in Genetics
and Development, Vol. 10, 193-198 (2000)
Lecture 1: Regulation of transcription in bacteria
A) The importance of low affinity binding for gene regulation
1. Multiple weak binding increases affinity in a synergistic manner
2. Allows for combinatorial control
3. Makes changes in binding affinity by protein modification more effective
4. Permits faster response times by increasing the rate of dissociation

Lecture 1: Regulation of transcription in bacteria (continued)
B) Structure of the E. coli RNA polymerase (Text pp 365-367)
1. Composed of 3 different subunits
2. Promoter specificity and binding determined by sigma class of
transcription factors (Text pp 430-432)
3. Promoter sequence defined by –10 and –35 elements which interact
with RNA polymerase in a topologically constrained mechanism (Text pp 226)
C) Mechanism of transcriptional initiation (Text pp 223-227)
1. Binding of RNA polymerase to promoter in a closed complex
2. Isomerization of bound polymerase to open complex
3. Release of sigma factor
4.. Role of pausing during elongation
D) Regulation of transcription (Text pp 417-420)
1. Promoter sequence affects both binding and isomerization
2. Transcription factors in bacteria are called repressors and activators
3. Regulation by occlusion of promoter sequence (Text pp418 and 420)
4. Transcription factors regulate binding affinity and kinetics of
isomerization
Lecture 2: The regulation of mRNA Stability in Eukaryotes
A) How important is mRNA stability for the regulation of gene expression
1) A mathematical model for mRNA decay
2) The time it takes for a cell to change the concentration of a mRNA from one
steady-state level to another only depends upon the rate of mRNA decay – it does
not depend on the rate of transcription!!!
B) Structural elements of eukaryotic mRNAs important for stability
1) The 5’ cap
2) The 3’ poly(A) tail
3) Stability determinants
C) Mechanism of mRNA decay
1) Processive exonucleolytic degradation
2) Which end goes first?
3) Regulation of mRNA turnover

DATES: 9/23/03, 9/24/03, 9/25/03 Lecturer: Joel Eissenberg, Ph.D.
Lecturers 7, 8, 9: Nucleic Acid Biology
In each lecture, I'll present some background material related to the key question of the
topic, then cover in some detail a research paper illustrating an experimental approach to
answering this question.
1. Chromatin structure and transcription I
Key question: How does a transcription factor interact with a nucleosomal template?
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
2. Chromatin structure and transcription II
Key question: How is chromatin modified to facilitate gene activation?
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
3. Chromatin structure and repression
Key question: How does a chromatin repressor silence genes?
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

DATES: 9/26, 2003, 9/29/03, 9/30/03 Lecturer: Dale Dorsett, Ph.D.
Lecturer 10: Nucleic Acid Biology
Recommended Text: Ptashne M, Gann A. Genes and Signals
Transcriptional Activation in Yeast: The Gal4 Paradigm
The Gal4 activator
Separate DNA-binding and activation domains
Activation domain structure
Squelching
Recruitment
Activator bypass
Nucleosomes and chromatin modifiers
Gal4 targets
Repression of Gal4 activation by Mig-1 and Tup
DNA looping and local concentration
Reading Assignment:
For both lectures of October 15 and 16, Chapters 1, 2 and 3 of Ptashne & Gann
Lecture 11: Activation in Higher Eukaryotes
Targets of recruitment
Recruitment at the Drosophila hsp70 gene
Combinatorial control and alternative enhancers
Remote enhancers
Insulators
DNA methylation and imprinting
Chromosomal position effects
Reading Assignment: see above
Lecture 12: RNA Processing
Production of Mature Messenger RNA and the mRNA Life Cycle
mRNA capping
mRNA splicing
reiterative splicing of long introns
mRNA polyadenylation
The PolII CTD and coordination of transcription and processing
mRNA transport
mRNA degradation and turnover
Degradation of missense mutant mRNA
Mechanisms of RNAi-induced degradation

Reading Assignment:
Bentley D. 1999. Coupling RNA polymerase II transcription with pre-mRNA
processing. Curr Opin Cell Biol 11:347-51
Hatton AR, Subramanian V, Lopez AJ. 1998. Generation of alternative
Ultrabithorax isoforms and stepwise removal of a large intron by resplicing at
exon-exon junctions. Mol Cell 2:787-96
DATES: 10/1/2003, 10/2/2003 Lecturer: Yie-Hwa Chang, Ph.D.
Lecturer 13: Nucleic Acid Biology
Protein synthesis I
Suggested readings:
Cell and Molecular Biology, Kleinsmith and Kish(2 nd edition), Chapter 11
Berg JM, Lorsch JR (2001) Mechanism of ribosomal peptide bond formation. Science.
291:203
Ibba, M., Soll, D. (1999) Quality control mechanism during translation. Science.
286:1893.
1. Ribosome structure
2. Protein synthesis
3. mRNA
4. tRNA
5. The initiation of protein synthesis
6. Peptide bond formation
7. Translocation
8. Termination of protein synthesis
9. Polysomes
OUTLINE

Suggested readings:
Lecture 14 : Protein synthesis II
Cell and Molecular Biology, Kleinsmith and Kish(2 nd edition), Chapter 11
Pestova TV, et al. (2001) Molecular mechanism of translation initiation in eukaryotes.
Proc. Natl. Acad. Sci. USA 98:7029.
Gale, M., Tan, S.L., Katze, M.G. (2000) Translational control of viral gene expression in
eukaryotes. Microbiol. Mol. Biol. Rev. 64:239.
1. Translational repressors
2. Life span of mRNA
3. Antisense RNA
4. Phosphorylation
5. Availability of tRNA
6. Rate of termination of transcription
7. Initiation factors
OUTLINE
DATE: 10/3/2003 Lecturer: Dorota Skowyra, Ph.D.
Lecture 15: Nucleic Acid Biology
Protein Folding and its Quality Control by Cellular Systems
Summary: I will briefly describe main intracellular protein folding machineries (molecular
chaperones, Hsp60, Hsp70) and introduce the issue of intracellular protein degradation,
highlighting the function of both systems in cellular protein quality control.
Introduction: 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 in the
time frame that is available. In the cell, protein folding is assisted by molecular
chaperones, which in multiple rounds of ATP-dependent binding, unfolding, and release
‘massage’ the protein into its optimal shape. If this process fails, the misfolded or
improperly assembled polypeptides are recognized by cellular quality control systems
and eliminated by targeted proteolysis . 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.
Problem (prepare your opinion to discuss in class): In your opinion, is the high rate of
degradation of the newly synthesized proteins a likely evolutionary solution to the
problem of optimizing the cost of a successful gene expression? What could be the
possible evolutionary advantage of the cellular ‘waste’ problem?
Recommended Reading:
(1). Alberts, B. et al. (1994): The Birth, Assembly and Death of Proteins, in: Molecular
Biology of the Cell, Chapter 5: Protein Function, pp. 212-222, Garland Publishing
(2). Wickner, S., Maurizi, M.R., Gottesman, S. (1999): “Post-translational Quality
Control: Folding, Refolding, and Degrading proteins.” Science 286, 1888-1893.
Advanced Reading (not required but suggested):
(3). Frydman, J. (2001): Folding of Newly Translated Proteins in vivo: The Role of
Molecular Chaperones.” Annu.Rev.Biochem. 70, 603-47
(4). Fewell, S.W., Travers, K.J., Weisman, J.S., Brodsky, J.L.: “ The Action of Molecular
Chaperones in the Early Secretory Pathway.” Annu.Rev.Genet. 35, 149-91
DATE: 10/6/03 Lecturer: Dorota Skowyra, Ph.D.
Lecture 16: Ubiquitination and Proteolysis as key Regulatory Mechanisms
of Gene Expression
Summary: I will provide an overview of the currently known mechanisms responsible for
substrate specificity in UPS (ubiquitin-proteasome system) and highlight their significance
in the regulation of gene expression.
Introduction: The current view is that the specificity and timing of intracellular proteolysis
are controlled at a step of assembly of polyubiquitin chains on the substrate protein. In
this view, the role of proteasomes is to recognize and degrade substrates that are selected
for degradation by one of the specific ubiquitination systems.
*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 extend and high 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 extra-cellular 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, temporary 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. Not surprisingly, defects in the ubiquitin-proteasome pathway have
been implicated in the pathogenesis of a broad range of human diseases, including several
forms of malignancies, a number of genetic diseases (cystic fibrosis, Angelman’s
syndrome, Parkinson’s disease, Liddle syndrome), immunological disorders, and muscle
wasting.
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?
Recommended Reading:
*(1). Selected chapters from: Glickman, M., and Ciechanover, A. (2002): “The
Ubiquitin-Proteasome Proteolytic Pathway: Destruction for the Sake of Construction”,
Physiol. Rev. 82: 373-428. Chapter I: Introduction and overview of ubiquitin-mediate
proteolysis. pp. 374- 376 Chapter II: The ubiquitin conjugating machinery: E1, E2, E3.
pp.377-381. Chapter IV: Modes of substrate recognition and regulation of the ubiquitin
pathway. pp. 383-388
DATE: 10/7/2003 Study Day
DATE: 10/8/2003 EXAM
9:00 – Noon LRC 105A, LRC 105B, LRC 106A
DATE: 10/9/03 4:00 pm Lecture Rm A – Medical School
Second William H. Elliott Lecture
“Beyond the Double Helix: Writing and Reading
the Histone Code”
presented by C. David Allis, Ph.D.
Joy and Jack Fishman Professor
Head, Laboratory of Chromatin Biology
The Rockefeller University, New York, NY

DATE: 10/10/03 Lecturer: John Tavis, Ph.D.
Lecture 18: Nucleic Acid Biology
Replication, Recombination and Repair
Note: Copies of all required reading material will be on reserve in the library
Oct. 10 (9:00) DNA Replication I
Topics: Necessity of DNA replication, biological and biochemical constraints on DNA
replication, overview of replication, initiation of DNA replication, enzymes of
replication, the replication fork.
Required reading: Alberts pp. 238-250, Stryer 799-809.
Oct. 10 (10:00) DNA Replication II
Topics: The replication fork in detail, termination and resolution, special problems for
eukaryotic replication, other modes of replication, fidelity of replication.
Required reading: Alberts pp. 251-266.
DATE: 10/13/03 Lecturer: John Tavis, Ph.D.
Lecture 19: DNA Repair I
Topics: Biological necessity for DNA repair, DNA damage, repair pathways overview,
photoreactivation.
Required reading: Alberts pp. 267-275.
DATE: 10/14/03 Lecturer: John Tavis, Ph.D.
Lecture 20: DNA Repair II
Topics: Base excision repair, nucleotide excision repair, SOS repair, recombinational
repair.
Required reading: Lindahl and Wood, Science 286:1897-1905.
DATE: 10/15/03 Lecturer: John Tavis, Ph.D.
Lecture 21: DNA Recombination I
Topics: Biological necessity for DNA recombination, homologous recombination
overview, synapsis, the Holliday junction.
Required reading: Alberts pp. 275-296.
DATE: 10/16/03 Lecturer: John Tavis, Ph.D.
Lecture 22: DNA Recombination II
Topics: The Holliday junction, outcomes of a Holliday junction, gene conversion,
recombination over double-stranded gaps, non-homologous recombination,
integration, and transposition.
Required reading: Cox, MM, et al. Nature 404:37-41.
Teaching Goals: The goal is to provide students with a basic understanding of the
biochemical mechanisms by which DNA is maintained in cells, the biological need for
fidelity in DNA replication, and how mutations and recombination lead to genetic
diversity. Focal points will be the DNA replication fork and the Holliday junction.

Textbooks:
Alberts, Johnson, Lewis, Raff, Roberts, and Walter. Molecular Biology of the Cell, 4 th
edition. 2002. Garland Science, Inc., New York. ISBN 0-8153-3218-1
Stryer. Biochemistry, 4 th edition. 1995. WH Freeman and Co., New York. ISBN 0-
7167-2009-4
10/17/03 EXAM
9:00 am to Noon Medical School – Auditorium A (first floor Medical School)