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Today…
Genome 351, 12 April 2013, Lecture 4
•mRNA splicing
•Promoter recognition
•Transcriptional regulation
•Mitosis: how the genetic material is partitioned
during cell division
In bacteria (most) mRNAs are co-linear
with their corresponding genes
Promoter
terminator
gene
AACTGACGA
+1
bacteria:
AACUGACGA
AACGA
mRNA
Events involved in RNA processing
Pre-mRNA
Intron
Exon1
Noncoding
Coding sequence
Noncoding
Exon2
Coding sequence
Noncoding
Noncoding
Continuous stretch of coding sequence
Noncoding
Noncoding
Continuous stretch of coding sequence
Noncoding
Transport to the cytoplasm
AAAAA
Why does transcript splicing occur?
Proteins can be
modular
-Different regions
can have distinct
functions
and the modules can
correspond to exons
Interrupted structure allows
genes to be modular
secretion
cell anchor
enzyme
binding module
Interrupted structure allows
genes to be modular
Pre-mRNA:
secretion
cell anchor
enzyme
binding module
secretion
cell anchor
enzyme
binding module
secretion
cell anchor
enzyme
binding module
Processed-mRNA
AAAA
Alternative splicing or:
One mRNAs exon is another one’s intron!
Pre-mRNA:
secretion
secretion
cell anchor
enzyme
enzyme
binding module
binding module
one alternative form
secretion
enzyme
binding module
AAAA
Processed-mRNA
Alternative splicing or:
One mRNAs exon is another one’s intron!
Pre-mRNA:
secretion
enzyme
enzyme
cell anchor
binding module
binding module
another alternative form
enzyme
binding module
AAAA
Processed-mRNA
How do RNA polymerases know where to
begin transcription and which way to go?
promoter
mRNA
mRNA
gene
promoter
gene
gene promoter
mRNA
First worked out in bacteria by:
-comparing sequences near the start sites of
transcription of many genes
-by studying where RNA polymerase likes to
bind to DNA
How do RNA polymerases know where to
begin transcription and which way to go?
Comparing sequences at the promoter region of many
bacterial genes provides clues:
direction of
transcription
only coding (sense) strand
transcription
start site
is shown; all sequences 5’-3’
-35 region
-10 region
consensus
sequence: TTGACAT…15-17bp…TATAAT
+1
RNA polymerase binds to the consensus
sequences in bacterial promoters
RNA polymerase binds to the -35 and -10 regions:
RNA
polymerase
direction of
transcription
TTGACAT
-35 region
TATAA
T
-10 region
+1
Would you expect RNA polymerase to bind the other
way around and transcribe in the reverse direction?
RNA polymerase binds to the consensus
sequences in bacterial promoters
RNA polymerase binds to the -35 and -10 regions:
RNA
polymerase
direction of
transcription
TTGACAT
-35 region
TATAA
T
-10 region
+1
Would you expect RNA polymerase to bind the other
way around and transcribe in the reverse direction?
RNA polymerase binds to the consensus
sequences in bacterial promoters
direction of
transcription
RNA
polymerase
TTGACAT
-35 region
TATAA
T
-10 region
+1
Would you expect RNA polymerase to bind this
sequence and initiate transcription?
T
T
direction of
transcription
AATA
T
ACAGTT
How do RNA polymerases know where to
begin transcription and which way to go?
In bacteria RNA polymerase binds specific sequences near
the start site of transcription that orient the polymerase:
mRNA
gene
mRNA
gene
TTGACAT
TATAAT
-35 region
-10 region
gene
mRNA
-10 region
-35 region
TAATAT
TACAGTT
In eukaryotes, RNA polymerase is
regulated by DNA-binding proteins
transcription factors (TF’s):
But TF’s that bind to
specific DNA sequences
& to RNA polymerase can
recruit RNA polymerase
& activate transcription
RNA polymerase:
+1
RNA polymerase does
not efficiently bind to
DNA and activate
transcription on its own
+1
In eukaryotes, RNA polymerase is
regulated by DNA-binding proteins
transcription factors (TF’s):
But TF’s that bind to
specific DNA sequences
& to RNA polymerase can
recruit RNA polymerase
& activate transcription
RNA polymerase:
+1
Some TF’s can also
inhibit transcription
+1
Switches and Regulators - A Metaphor
• Switches control transcription (which take the form of
DNA sequence)
- Called regulatory elements (RE’s) or enhancers
- Adjoin the promoter region, but can be quite distant
• Regulators, which take the form of proteins that bind the
DNA, operate the switches
- Called transcription factors (TF’s)
• When and how much RNA is made often is the product of
multiple elements and regulators
Control of gene expression
•Each cell contains the same genetic
blueprint
•Cell types differ in their protein
content
•Some genes are used in almost all
cells (housekeeping genes)
•Other genes are used selectively in
different cell types or in response to
different conditions.
An imaginary regulatory region
RE1
RE2
RE3
RE4
RE5
RE6
Promoter
Expressing a regulatory gene in the wrong
place can have disastrous consequences!!!
Example: Antennapedia gene in fruit flies
Antennapedia gene is
normally only transcribed in
the thorax; legs are made.
A mutant promoter causes the
Antennapedia gene to be
expressed in the thorax and
also in the head, where legs
result instead of antennae!
Lactose tolerance: A human example of a
promoter mutation
Lactase levels
Lactase levels in humans
2
10
Age in years
World wide distribution of
lactose intolerance
The cellular life cycle
Mitosis: dividing the
content of a cell
fertilized egg;
a single cell!
Chromosomes - a reminder
How many do humans have? •22 pairs of autosomes
•2 sex chromosomes
•Each parent contributes
one chromosome to each
pair
•Chromosomes of the
same pair are called
homologs
•Others are called nonhomologous
Photo: David McDonald, Laboratory of
Pathology of Seattle
Homologous and non-homologous chromosomes
The zygote receives
one paternal (p) and
one maternal (m) copy
of each homologous
chromosome
1p
2p
3p
1m
2m
3m
21p
22p
Xp or Y
21m
22m
Xm
The DNA of human chromosomes
# base pairs # genes
# base pairs # genes
The cellular life cycle
Elements of mitosis:
cell growth;
chromosome
duplication
cell growth;
chromosome
duplication
chromosomes
decondensed
chromosome
segregation
chromosomes
condensed
chromosome
segregation
repeat
Chromosome replication – a
reminder
• Mechanism of DNA
synthesis ensure that
each double stranded
DNA gets copied only
once.
• The products of DNA
replication have one new
DNA strand and one old
one (semi-conservative
replication)
Chromosome structure – a reminder
chromosome structure
during cell growth &
chromosome replication
(decondensed)
held together at
the centromere
sister chromatids; doublestranded DNA copies of
the SAME homolog
Mitosis -- making sure each daughter cell
gets one copy of each pair of chromosomes
• Copied chromosomes
(sister chromatids)
stay joined together at
the centromere.
• Proteins pull the two
sister chromatids to
opposite poles
• Each daughter cell gets
one copy of each
homolog.
Mitosis -- homologous chromosomes
1m
1p
2 copies 1m
2 copies 1p
2 copies 1m
2 copies 1p
1m
1p
1m
1p
1m
1p
1m
1p
exact copies
joined at
centromer
e
Mitosis – following the fate of CFTR
CFTR+
2 copies CFTR+
2 copies CFTR+
CFTR+
CFTRCFTR+
CFTR-
CFTR2 copies CFTR2 copies CFTRCFTR+
CFTRCFTR+
CFTR-
A CFTR
heterozygote
(CFTR+/CFTR-)
A closer look at the chromosomes
Paternal chromosome
CTCCTCAGGAGTCAGGTGCAC
GTGCACCTGACTCCTGAGGAG
CTCCACAGGAGTCAGGTGCAC
GTGCACCTGACTCCTGTGGAG
Maternal chromosome
Mitosis -- 2 copies of each chromosome at the start
A closer look at the chromosomes
CTCCTCAGGAGTCAGGTGCAC
GTGCACCTGACTCCTGAGGAG
CTCCACAGGAGTCAGGTGCAC
GTGCACCTGACTCCTGTGGAG
DNA strands separate followed
by new strand synthesis
A closer look at the chromosomes
CTCCTCAGGAGTCAGGTGCAC
GTGCACCTGACTCCTGAGGAG
CTCCTCAGGAGTCAGGTGCAC
GTGCACCTGACTCCTGAGGAG
CTCCACAGGAGTCAGGTGCAC
GTGCACCTGACTCCTGTGGAG
CTCCACAGGAGTCAGGTGCAC
GTGCACCTGACTCCTGTGGAG
• Mitosis -- after replication 4
copies
• Homologs unpaired sister
chromatids joined by centromere
A closer look at the chromosomes
CTCCTCAGGAGTCAGGTGCAC
GTGCACCTGACTCCTGAGGAG
CTCCACAGGAGTCAGGTGCAC
GTGCACCTGACTCCTGTGGAG
CTCCTCAGGAGTCAGGTGCAC
GTGCACCTGACTCCTGAGGAG
CTCCACAGGAGTCAGGTGCAC
GTGCACCTGACTCCTGTGGAG
Each daughter has a
copy of each homolog
Mitosis and the cell cycle
Mitosis vs. Meiosis
- The goal of mitosis is to
make more “somatic” cells:
each daughter cell should
have the same chromosome
set as the parental cell
- The goal of meiosis is to
make sperm and eggs:
each daughter cell should
have half the number of
chromosome sets as the
parental cell
Meiosis: the formation of gametes
The challenge:
• ensuring that homologues
are partitioned to
separate gametes
The solution:
• Hold homologous
chromosomes together by
crossing over
• target homologues to
opposite poles of the cell…
• then separate the
homologues