Download Lecture#7 - Eukaryote gene structure and regulation.

5/6/17
BIOLOGY 207 - Dr. Locke
Lecture#7 - Eukaryote gene structure and regulation.
Required readings and problems:
Reading: Open Genetics, Chapter 12
Problems: Chapter 12
Optional
Griffiths (2008) 9th Ed. Readings: pp 33-36, 385-402; 300-312
Problems: 9th Ed. Ch. 8: 2,3,6,8,12-14,18 Ch 11: 1,2,5,16
Campbell (2008) 9th Ed. Readings: Concept 17.1-3, 18.2
Concepts:
How are eukaryote genes organized & regulated?
1. Higher Eukaryote genes are generally larger and more complex than those of
Prokaryotes.
2. Their transcripts are processed in the nucleus before being exported to the
cytoplasm, where they are translated.
3. Proteins interact with various DNA sequences to regulate gene expression.
4. Eukaryotic genes are regulated at the promoter, by enhancers/silencers, and by
chromatin structure, too.
Biol207 Dr. Locke section
Lecture#7
Fall'11
page 1
5/6/17
The eukaryote genome contains various different types of
sequences
Types of sequences
1) Single copy genes - includes most protein coding genes
2) Multiple copy genes - e.g. rRNA, tRNA, some protein coding genes.
3) Repeated sequences.
3a Short centromeric - tandem array - clustered
3b SINES Short INterspersed ElementS (Alu element – lab #9)
3c LINES Long INterspersed ElementS
3d VNTR Variable Number Tandem Repeats
Higher Eukaryotes have an excess of DNA
- more than needed to code for protein encoding genes.
- rest -> ???? - C-value paradox Junk DNA?
-----------------------------------------O-------------------------------------
Biol207 Dr. Locke section
Lecture#7
Fall'11
page 2
5/6/17
Transcripts in Eukaryotes:
Monocistronic and modified
Steps in the processing of Eukaryote mRNA
a. Transcript produced by RNA polymerase II (for mRNA)
b. Addition of 7-methyl guanosine cap to 5' end
c. Transcription past poly(A) site
d. Endonuclease cleavage after AAUAAA signal sequence.
e. Addition of poly(A) tail as series of 150-200 A by poly(A) polymerase
f. Complete primary mRNA - not yet
"mature"
Introns and exons:
Primary transcripts are shortened by
removal of intervening sequences
(introns) before transport to the
cytoplasm.
Fig
Biol207 Dr. Locke section
Lecture#7
Fall'11
page 3
5/6/17
Example: the ovalbumin gene/mRNA in chickens.
1- Primary transcript of ~7,700 bp has 5' cap and 3' poly A added,
2- The 7 introns (A-G) spliced out in a series of steps to produce a mature mRNA of
1872 nucleotides.
3- Regions L and 1-7 are called exons (vs. introns) and are joined together to form the
mature mRNA.
The biochemistry of intron splicing is well understood and involves the lariat model.
For some genes (most) the processed mature mRNA is the same product each time.
Biol207 Dr. Locke section
Lecture#7
Fall'11
page 4
5/6/17
Alternative Splicing
Some genes have primary transcripts that are processed to produce more than one
type of mature mRNA - > more than one type of protein product
The same primary transcript can be processed to yield different mRNAs, which encode
different proteins.
(Another means of gene regulation - post-transcriptional level)
Example:
Fig Rat -tropomyosin gene
Note:
1- Tissue specific differences in expression
2- Alternate 5’ (transcription initiation sites)
3- Alternate 3’ ends ( poly A sites)
4- What does intron/exon mean?
5- Different genes have different levels of alternate splicing
Biol207 Dr. Locke section
Lecture#7
Fall'11
page 5
5/6/17
Eukaryote Regulation of Transcription
In prokaryote genes:
- positive and negative regulators that involve
trans-acting factors (e.g. I gene-->lac operon) that act on
cis-dominant regulatory sequences (e.g. operators or initiators, or binding sites
in the promoter).
In eukaryote genes:
- also have trans-acting factors and cis-acting regulatory sequences
Proximal cis-acting elements - The Promoter Region
Fig beta-globin promoter
Mutations have been used to define the essential promoter sequences.
Many nucleotides can be changed with no effect on transcription.
DNA sequences in the promoter region of the gene:
|-----> mRNA
- - - - GGGCGG- - - - - CCAAT - - - - - - - TATA - - - - - - - - - - - - - - - 110
-40
- 30
1
Three sequences are frequently found immediately upstream from the mRNA
transcription start site.
"TATA box" sequence serves to guide the RNA polymerase to begin transcription
about 30 bp downstream at the start site.
Biol207 Dr. Locke section
Lecture#7
Fall'11
page 6
5/6/17
Distal elements - Enhancer sequences /silencers
Enhancers (upstream activating sequences - UAS in yeast) are cis-acting sequences
that can increase rates of transcription from an adjacent promoter on the same
molecule (or chromosome).
They can act at distances of many Kbp - upstream or downstream from the promoter
that they affect
several Kb
|----> mRNA
- - - - - - Enhancer - - - - - - - - - Promoter - - - - - - Example:
Yeast Gal4 activation
Fig
Gal4- dimer - separate DNA binding and activation domains
Binds to an enhancer to positively influence the adjacent genes promoter and
increases transcription.
Transcriptional activator proteins recuit the transcriptional machinery.
Fig
Bring RNA Pol II & other proteins to promoter to increase transcription.
Enhancers, promoters and transacting factors, together, regulate transcription.
Biol207 Dr. Locke section
Lecture#7
Fall'11
page 7
5/6/17
Chromatin regulates gene expression
Euchromatin
- loosely packaged, accessible by transcription enzymes
- gene expression is possible (but not obligatory)
Heterochromatin
- tightly packaged, inaccessible by transcription enzymes
- gene expression is not possible (not permitted)
Chromosome specific heterochromatinization.
Human X-chromosome
MALE :
Y/X one X 1 copy of genes
FEMALE: X/X two X 2 copies of genes
-> need to compensate -> dosage compensation
- 1 copy worth by X-chromosome inactivation (random)
- X-chromosome wide repression
- no genes expressed (almost none)
- chromatin specific to that X is responsible.
Biol207 Dr. Locke section
Lecture#7
Fall'11
page 8
5/6/17
Inactivated chromatin structure of the
X-chromosome is inherited in the daughter cells (epigenetics)
Fig
Both XM and XP are present in each female cell but one is inactivated during early
development
This random event becomes an inherited property of that chromosome, and all its
daughter chromosomes are inactivated, too, even the cell is put into in vitro
culture.
Biol207 Dr. Locke section
Lecture#7
Fall'11
page 9