Transcript Why don’t antibodies get rid of HIV?

Signals in DNA tell RNA polymerase
where to start and stop transcription
• Synthesis starts at a promoter,
a conserved sequence 5’ of a gene.
• Chain elongation occurs until
RNA polymerase encounters a
termination sequence on the DNA.
• RNA polymerase releases the
single stranded RNA and the
double stranded DNA template
upon encountering a termination
sequence.
• This slide depicts a bacterial
RNA polymerase. Transcription
initiation in eukaryotic cells is
more complicated.
Promoter and terminator sequences in
bacteria
Clicker question
Propose a mechanism by which the highlighted areas
could cause transcription to stop.
5)
The first two sequences are inverted repeats of each other, thus a hairpin,
which is a signal for termination.
What is the same in all* somatic cells
of your body?
1)
2)
3)
4)
5)
6)
DNA
RNA
Proteins
Lipids
Carbohydrates
Lysosomes
* Some immune cells are interesting exceptions.
The DNA in differentiated cells contains all
the instructions to make a new organism
This is reproductive cloning -- how Dolly the
Sheep was created. In theory, this procedure
could be done in humans, but it would be
unethical because most of the embryos are
abnormal.
• Cell types differentiate during development of a multicellular organism to become
specialized (e.g., muscle, nerve, brain, blood cells are different from each other), but
they contain the same DNA.
• What is different is that they express different genes, so they accumulate
different sets of RNA and protein.
Cells regulate expression of proteins at
many levels
We will discuss transcriptional control.
Transcription is controlled by proteins
binding to regulatory sequences on DNA
• Almost all prokaryotic and eukaryotic genes have
regulatory DNA sequences that are used to switch
the gene on or off.
• Gene regulatory proteins bind to the regulatory
DNA sequences.
- Repressors prevent transcription
- Activators promote transcription
• Early studies of gene regulation involved
bacteriophage and bacterial genes, which have
simpler regulatory regions than eukaryotic genes.
Repressors block RNA
polymerase -- prevent
transcription initiation
Activators interact with
RNA polymerase to help
initiate transcription
Bacteriophages (bacteria eaters) -Viruses that infect bacteria
• After infection, phage can live in one of two
states
– Lytic: phage degrades host DNA, hijacks host cell
machinery to produce many new phage. When cell is
depleted, phage lyse their host bacterium to release
new phage.
– Lysogenic: phage DNA integrates into host
chromosome. Prophage (phage DNA) duplicated every
time host divides. Stress to host causes excision of
prophage DNA and entry into lytic phase.
• Classic example of transcriptional regulation
to turn on and off genes
Viruses that infect bacteria (bacteriophage or phage) look
like syringes
Levine, A. “Viruses” p. 34
Bacteriophage infection
www.seyet.com/t4_academic.html
Growth of phage 
Figure 1.2. Mark Ptashne. A Genetic Switch: Gene Control and Phage 
Genetic switch between two states in
phage 
(cI protein
also called
 repressor)
Lysogenic
state
Lysogenic state
Lysogenic genes on; lytic genes off
repressor (cI protein) blocks transcription of Cro
(a lytic gene) and activates transcription of its own gene.
 Repressor
gene is on
Phage genes are off
A repressor physically blocks RNA pol
from initiating transcription.
An activator interacts with RNA pol
to promote transcription.
 ”repressor” acts as both a repressor and an activator
Lytic state
Lytic genes on; lysogenic genes off
Cro repressor binds to OR3, preventing transcription of
 repressor (cI protein) and other lysogenic genes. RNA
pol can now transcribe Cro gene.
 Repressor
gene is off
Phage genes are on
A genetic switch in prokaryotes (lysis versus lysogeny of a
virus) is controlled by regulation of transcription
Repressor
gene is on
Repressor
gene is off
Phage genes are off
Phage genes are on
Lysogenic state: Bacteriophage
genome is integrated into
bacterial genome. Bacteriophage
is dormant and phage genes are
not expressed. Viral repressor
proteins (red dumbbells) block
transcription of lytic genes and
activate transcription of
repressor genes.
Lytic state: UV light or other
inducer causes phage genes to be
turned on and new phage to be
produced, which results in lysis
of the host cell. Viral repressors
(green spheres) prevent
transcription of lysogenic genes,
but not lytic genes.
Transcriptional regulators (repressors, activators) are
proteins that recognize specific DNA sequences
• a-helices of
proteins fit into the
major groove of
DNA.
• Amino acid
sidechains from the
protein make
specific contacts
with exposed edges
of basepairs.
Structure of bacteriophage lambda repressor
Beamer & Pabo, 1992, J. Mol. Biol. 227: 177.
Sequence-specific recognition of DNA by proteins
Seeman et al. (1976) PNAS 73:804-808
A
A
D
Major groove
Major
groove
Minor
groove
Minor groove
A
D
A
Clicker question
1)
2)
3)
4)
5)
6)
Many DNA binding proteins (including restriction
enzymes) recognize palindromic* sequences of
DNA. What does binding to a palindromic DNA
sequence imply about the structure of a DNAbinding protein?
It is a dimer with translational symmetry
It is a dimer with two-fold rotational symmetry
It is a dimer with mirror (inversion) symmetry
It is a trimer with translational symmetry
It is a trimer with three-fold rotational symmetry
It is a trimer with three-fold mirror symmetry
*Palindrome examples: MADAM I’M ADAM or
GAAGCTCGTACGAGCTTC
CTTCGAGCATGCTCGAAG
Clicker question
What does binding to a palindromic DNA
sequence imply about the structure of the DNAbinding protein?
1) It is a dimer with translational symmetry
2) It is a dimer with two-fold rotational symmetry
3) It is a dimer with two-fold mirror symmetry
*Palindrome examples: MADAM I’M ADAM or
GAAGCTCGTACGAGCTTC
CTTCGAGCATGCTCGAAG
Transcriptional regulation of eukaryotic genes is more
complicated than regulation of prokaryotic genes
• Basal promoter elements (e.g., TATA box: 25 bp
upstream of transcription start site).
• Promoter proximal element. 100-200 bp long -- close to
site of transcription initiation. Contains sequences
recognized by different transcription factors.
• Enhancer elements. Can be a few thousand to 20,000 bp
upstream or downstream from the initiator site.
Transcriptional activation involves interactions over
long stretches of DNA
TBP
•
TATA box binding protein (TBP) binds to
RNA polymerase and other proteins to
form pre-initiation complex.
TATA-binding protein (TBP)
Transcriptional activation involves interactions over
long stretches of DNA
•
•
•
TATA box binding protein (TBP) binds to RNA pol and other proteins to
form pre-initiation complex.
Pre-initiation complex interacts with different specific transcription
factors bound to promoter proximal elements and enhancer elements.
Each gene in every cell has same DNA control sequences, but not every
cell has complete set of DNA binding proteins to turn on every gene.
NFkB
Stress signal
comes from
outside the cell.
A signaling
cascade in the
cytoplasm results
in degradation of
I-kB.
NFkB enters
the nucleus
and activates
genes.
• NFkB proteins are cytoplasmic.
Inactive when bound to I-kB.
• Inducers (stress, infection, etc.)
trigger dissociation and
degradation of I-kB, then NFkB
enters nucleus and activates
genes.
• NFkB binds to kB sites in the
enhancer regions of genes
involved in cellular defense
mechanisms and differentiation.
• NFkB induces transcription of
HIV viral RNA by the host cell
RNA polymerase.
• David Baltimore’s lab at Caltech
works on NFkB.
Crystal structure a piece of NFkB bound to a kB site on DNA
•
•
Structure resembles butterfly with protein domains as wings attached to
cylindrical body of DNA.
Contacts with DNA formed by loops between b-strands. No helical or
sheet structure at recognition surface.
Ghosh et al., 1995, Nature 373: 303-310; Müller et al., ibid, 311-317
How can you tell when/where a gene is
being expressed?
Hybridization can be used to locate when/where a
gene is expressed or compare genomes
•
•
•
Take advantage of ability of DNA to pair selectively with a second
strand of complementary nucleotide sequence.
Denature DNA (heat or pH) to separate strands, then slowly
reverse to allow double helices to reform (hybridization or
renaturation).
Any two complementary single-stranded nucleic acid chains can
hybridize (DNA/DNA, RNA/RNA, RNA/DNA). [HIV reverse
transcriptase makes cDNA, or complementary DNA, from a singlestranded RNA template.]
Figure 10-12, Little Alberts
Physical Chemistry of DNA Hybridization
Studied at Caltech in 60s and 70s
by Norman Davidson
1. The hydrogen bonds that
form double-stranded DNA are
easily disrupted by heating.
2. Some dyes fluoresce when
they bind to double-stranded
DNA.
Can use hybridization to search for gene expression
• Make a ssDNA probe (can be synthesized) to detect
complementary sequences of interest.
• Can probe for normal versus mutant forms of a gene (e.g., sicklecell anemia gene; cancer-predisposing genes).
• Can probe cDNA from cells at a particular developmental stage
or from a tissue to see when or where a gene is expressed.
• Can do hybridizations in situ (in place) to locate nucleic acid
sequences in cells, organisms (below left) or on chromosomes
(below right).
FISH -- Fluorescence
In Situ Hybridization
Whole mount zebrafish in situ hybrization kit
2 mm
DNA microarrays to evaluate gene
expression
• Array thousands of DNA oligonucleotides (probes)
in known locations, each is a different sequence
• Add target cDNA (complementary DNA made
from transcribed RNA) (target) to hybridize under
high-stringency conditions
• Probe-target hybridization detected by and
quantified by fluorescence
Commercial Gene Chips
Probes attached covalently to a chemical matrix on a solid surface (quartz). Made using
photolithography.
Applications
• Gene expression profiling -- monitor mRNA for
thousands of genes to study effects of:
– different stages in development or differentiation
– disease (cancer)
– infection
• Compare genomes in different organisms
• Identify single nucleotide polymorphisms (SNPs)
for genotyping, forensics, to study predisposition
to disease, find mutations in cancers
• Alternative splicing detection -- use probes for
predicted exons of a gene
Dual Color
Microarray
experiment
Red Fluorescent Probes
Green Fluorescent Probes
n.m.wikipedia.org/wiki/DNA_microarray
~40,000 probe spotted oligo microarray
Yellow -- merge of red and green fluorescence
n.m.wikipedia.org/wiki/DNA_microarray