Today… Genome 351, 12 April 2013, Lecture 4 mRNA splicing Promoter recognition Transcriptional...
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Transcript of Today… Genome 351, 12 April 2013, Lecture 4 mRNA splicing Promoter recognition Transcriptional...
Today…Genome 351, 12 April 2013, Lecture 4
•mRNA splicing
• Promoter recognition
• Transcriptional regulation
•Mitosis: how the genetic material is partitioned during cell division
Please be sure to turn in your first problem set assignment today, and also pick up the
second problem sethttp://courses.washington.edu/gen351/
In bacteria (most) mRNAs are co-linear with their corresponding genes
+1
Promoter terminator
bacteria:(pre-mRNA)
(processed mRNA)
eukaryotes:
introns
exons
AACUGACGA
AACTGACGA
mRNA
AACGA
gene
Transcription
Translation
introns are removed during transcription in the nucleus
Events involved in RNA processing
Non-coding
Non-coding
Coding sequence Coding sequenceNoncoding
Exon1 Exon2Intron
Non-coding
Non-coding
Continuous stretch of coding sequence AAAAA
Add a string of A’s to the end
Non-coding
Non-coding
Continuous stretch of coding sequence
Intron removedSplice out the intron
Transport to the cytoplasm
Pre-mRNA
Processed-mRNA
Proteins can be modular-Different regions can have distinct functionsand the modules can correspond to exons
Why does transcript splicing occur?
Interrupted structure allows genes to be modular
secretion cell anchor enzyme binding module
introns
exons
introns can also lie in untranslated sequences
untranslated sequences
Interrupted structure allows genes to be modular
secretion cell anchor enzyme binding module
introns
exons
introns can also lie in untranslated sequences
untranslated sequencesPre-mRNA:
secretion cell anchor enzyme binding module
secretion cell anchor enzyme binding module
Pre-mRNA:
Processed-mRNA
Interrupted structure allows genes to be modular
secretion cell anchor enzyme binding module AAAA
This form stays anchored to the plasma membrane
Three introns removed
polyA tail added
secretion cell anchor enzyme binding module
secretion enzyme binding module
Pre-mRNA:
Processed-mRNA
Alternative splicing or:One mRNAs exon is another one’s
intron!
AAAAsecretion enzyme binding module
This form is secreted
Three introns & an exon removed
one alternative form
secretion cell anchor enzyme binding module
enzyme binding module
Pre-mRNA:
Processed-mRNA
Alternative splicing or:One mRNAs exon is another one’s
intron!
AAAAenzyme binding module
This form is retained in the cytoplasm
Three introns & two exons removed
another alternative form
Many additional possibilities with alternative splicing
New York Times
ApoE gene
Promoter
exons
cys cys
112 158
cys arg
arg arg
ApoE2
ApoE3
ApoE4
112 158Allele
10-30-fold increased risk of AD
8%
78%
14%
worldwidefrequency
How do RNA polymerases know where to begin transcription and which way to go?
promoter
mRNA
mRNA
promotergene
genegenemRNA
promoter
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
Comparing sequences at the promoter region of many bacterial genes provides clues:
How do RNA polymerases know where to begin transcription and which way to go?
consensussequence: TTGACAT…15-17bp…TATAAT
transcription start site
direction of transcriptio
n
+1-10 region-35 regionare these important
?
only coding (sense) strand is shown; all sequences 5’-3’
Promoter Strength (# of mRNAs
made/time)
Relatedness of promoter to consensus
TTGACAT…15-17bp…TATAAT -35 -10
ACAGTGA…15-17bp…CTGTCA -35 -10
RNA polymerase binds to the consensus sequences in bacterial promotersRNA polymerase binds to the -35 and -10 regions:
+1-10 region-35 region
TATAAT
direction of transcriptio
n
Would you expect RNA polymerase to bind the other way around and transcribe in the reverse direction?
TTGACAT
RNA polymerase
-35 binding part of RNA polymerase
-10 binding part of RNA polymerase
RNA polymerase binds to the consensus sequences in bacterial promotersRNA polymerase binds to the -35 and -10 regions:
+1-10 region-35 region
TATAAT
direction of transcriptio
n
Would you expect RNA polymerase to bind the other way around and transcribe in the reverse direction?
TTGACAT
RNA polymerase
-10 binding part of RNA polymerase
-35 binding part of RNA polymerase
RNA polymerase binds to the consensus sequences in bacterial promoters
+1-10 region-35 region
TATAATTTGACAT
RNA polymerase
direction of transcriptio
n
RNA polymerase
TAATATTACAGTT
direction of transcriptio
n
Would you expect RNA polymerase to bind this sequence and initiate transcription?
5’3’
5’ 3’
5’ TTGACAT 3’ 3’ TTGACAT 5’=These are chemically distinct molecules with different 3-D shapes!!
mRNA
mRNAgene
genegenemRNA
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:
-10 region-35 region
TTGACAT TATAAT
-35 region-10 region
TACAGTTTAATAT
similar principles- but a different mechanism-orients RNA polymerase in eukaryotes
In eukaryotes, RNA polymerase is regulated by DNA-binding proteins
RNA polymerase:
+1RNA polymerase does not efficiently bind to DNA and activate transcription on its own
In eukaryotes, RNA polymerase is regulated by DNA-binding proteins
RNA polymerase:transcription factors (TF’s):
+1RNA polymerase does not efficiently bind to DNA and activate transcription on its own
+1
But TF’s that bind to specific DNA sequences & to RNA polymerase can recruit RNA polymerase & activate transcription
In eukaryotes, RNA polymerase is regulated by DNA-binding proteins
RNA polymerase:
Some TF’s can also inhibit transcription
transcription factors (TF’s):
+1
+1
But TF’s that bind to specific DNA sequences & to RNA polymerase can recruit RNA polymerase & activate transcription
RNA polymerase does not efficiently bind to DNA and activate transcription on its own
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
Promoter
RE1 RE2 RE3 RE4 RE5 RE6
Controls timing of transcription
Inhibits transcription
Increases transcription
Turns on in brain
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!
Example: Antennapedia gene in fruit flies
Expressing a regulatory gene in the wrong place can have disastrous consequences!!!
Lactose tolerance: A human example of a promoter mutation
Lactase levels in humansLa
ctase
levels
Age in years2 10
lactose intolerant
lactose tolerant
World wide distribution of lactose intolerance
Convergent evolution: independent acquisition of the same biological trait in distinct populations
The cellular life cycle
fertilized egg; a single cell!
How is the genetic material equally divided during
mitosis?
The formation of sperm and eggs-more later on this subject
Mitosis: dividing the content of a
cell
Chromosomes - a reminder
How many do humans have?
Photo: David McDonald, Laboratory of Pathology of Seattle
• 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 non-homologous
Homologous and non-homologous chromosomes
1p 1m
2p 2m
3p 3m
21p
22m22p
21m
Xp or Y Xm
homologous
non-homologous
The zygote receives one paternal (p)
and one maternal (m) copy of each
homologous chromosome
homologous
The DNA of human chromosomes
# genes# base pairs # genes# base pairs
The cellular life cycle
cell growth; chromosome duplication
chromosomes
decondensed
cell growth; chromosome duplication
Elements of mitosis:
What are decondensed chromosomes?
How are chromosomes duplicated?
Chromosome structure – a reminder
chromosome structure during cell growth & chromosome replication (decondensed)
a condensed chromosome
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)
The cellular life cycle
cell growth; chromosome duplication
chromosome segregationcell growth;
chromosome duplication
chromosomes
decondensedchromosome segregation
chromosomes condensed
repeat
Elements of mitosis:
only showing a single duplicated homolog – 45 others not shown
Chromosome structure – a reminder
chromosome structure during cell growth & chromosome replication (decondensed)
a condensed chromosome
sister chromatids; double-stranded DNA copies of the SAME homolog
held together at the centromere
The cellular life cycle
cell growth; chromosome duplication
chromosome segregationcell growth;
chromosome duplication
chromosomes
decondensedchromosome segregation
chromosomes condensed
repeat
Elements of mitosis:
only showing a single duplicated homolog – 45 others not shown
The cellular life cycle
cell growth; chromosome duplication
chromosome segregationcell growth;
chromosome duplication
chromosomes
decondensedchromosome segregation
chromosomes condensed
repeat
Elements of mitosis:
Mitosis -- making sure each daughter cell gets one copy of each pair of
chromosomes
Understand what’s happening to the 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
1m
1p1m
1p
joined at centromere
2 copies 1m
1m
1p1m
1p
2 copies 1p
exact copies
Mitosis – following the fate of CFTR
1m 1p
2 copies 1m 2 copies 1p
1m
1p1m
1p
joined at centromere
2 copies 1m
1m
1p1m
1p
2 copies 1p
exact copies
Mitosis – following the fate of CFTR
CFTR+
CFTR-CFTR+
CFTR-
2 copies CFTR+ 2 copies CFTR-
2 copies CFTR+
2 copies CFTR-
CFTR+
CFTR-CFTR+
CFTR-
exact copies
CFTR+ CFTR-
A CFTR heterozygote (CFTR+/CFTR-)
GTGCACCTGACTCCTGAGGAG
CTCCTCAGGAGTCAGGTGCAC
GTGCACCTGACTCCTGTGGAG
CTCCACAGGAGTCAGGTGCAC
Mitosis -- 2 copies of each chromosome at the start
Paternal chromosome
Maternal chromosome
A closer look at the chromosomes
GTGCACCTGACTCCTGAGGAG
CTCCACAGGAGTCAGGTGCAC
CTCCTCAGGAGTCAGGTGCAC
GTGCACCTGACTCCTGTGGAG
DNA strands separate followed by new strand synthesis
A closer look at the chromosomes
GTGCACCTGACTCCTGAGGAG
CTCCTCAGGAGTCAGGTGCAC
GTGCACCTGACTCCTGTGGAGCTCCACAGGAGTCAGGTGCAC
GTGCACCTGACTCCTGTGGAG
CTCCACAGGAGTCAGGTGCAC
• Mitosis -- after replication 4 copies
• Homologs unpaired; sister chromatids joined by centromere
GTGCACCTGACTCCTGAGGAG
CTCCTCAGGAGTCAGGTGCAC
A closer look at the chromosomes
GTGCACCTGACTCCTGAGGAG
CTCCTCAGGAGTCAGGTGCACGTGCACCTGACTCCTGTGGAG
CTCCACAGGAGTCAGGTGCAC
GTGCACCTGACTCCTGTGGAG
CTCCACAGGAGTCAGGTGCAC
Each daughter has a copy of each homolog
GTGCACCTGACTCCTGAGGAG
CTCCTCAGGAGTCAGGTGCAC
A closer look at the chromosomes
Mitosis and the cell cycle
DNA synthesis
Chromosome condensation
Chromosome alignment
One copy of each chromosome to each daughter
Nuclear membrane breakdown
Mitosis vs. Meiosis
Meiosis: the formation of gametes
Mitosis: dividing somatic cells
- 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
number of copies of any given chromosome/cell (n):
number of copies of any given chromosome/sperm or egg:
2
1
1n
1n
2n 2n
2n 2n
2n 2n
4n!!
2n 2n
1n 1n
2nzygote:
Why reduce the number of chromosome sets during meiosis?
2n = diploid
1n = haploid
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 homologues3 other combinations
possible!
1n 1n
2n 2n