Multiple levels of eukaryotic gene expression and regulation

Genomic Control
DNA Rearrangement: eg of gene regulation
Cassette Mechanism of the yeast mating-type switch
Chr 3 has 3 copies of mating type information. MAT locus contains alpha- or a- specific DNA
and specifies mating type.
HMRa and HML alpha: They are transcriptionally silenced by SIR genes.
When alpha or a cell switches mating types the alpha or a DNA at the MAT locus is excised
and a DNA cassette copy of alternating mating-type is inserted.
Cells frequently switch mating type, as a means of maximizing opportunities for mating.
The yeast Saccharomyces cerevisiae is a simple single celled eukaryote with
both a diploid and haploid mode of existence. The mating of yeast only
occurs between haploids, which can be either the a or (alpha) mating type
and thus display simple sexual differentiation. Mating type is determined by a
single locus, MAT, which in turn governs the sexual behaviour of both haploid
and diploid cells.

S. cerevisiae (yeast) can stably exist as either a diploid or a haploid. Both
haploid and diploid yeast cells reproduce by mitosis, with daughter cells
budding off of mother cells. Haploid cells are capable of mating with other
haploid cells of the opposite mating type (an a cell can only mate with an
cell, and vice versa) to produce a stable diploid cell. Diploid cells, usually
upon facing stressful conditions such as nutrient depletion, can undergo
meiosis to produce four haploid spores: two a spores and two spores.

Two haploid yeast of opposite mating types secrete pheromones, grow projections and mate.

a cells produce a-factor, a mating pheromone which signals the presence of an a cell to
neighboring cells. Vice versa also.
The different sets of transcriptional repression and activation which characterize a and cells
are caused by the presence of one of two alleles of a locus called MAT: MATa or MAT. The
alleles present at the MAT locus are sufficient to program the mating behaviour of the cell.
HML and HMR: the silent mating cassettes
Haploid yeast switch mating type by replacing the information present at the
MAT locus. For example, an a cell will switch to an cell by replacing the MATa
allele with the MAT allele. This replacement of one allele of MAT for the other is
possible because yeast cells carry an additional silenced copy of both the MATa
and MAT alleles: the HML (Hidden MAT Left) locus typically carries a silenced
copy of the MAT allele, and the HMR (Hidden MAT Right) locus typically carries
a silenced copy of the MATa allele. The silent HML and HMR loci are often
referred to as the silent mating cassettes, as the information present there is
'read into' the active MAT locus.
Location of the silent HML and HMR loci and
the active MAT locus on yeast chromosome III.
Mechanics of the mating type switch
The process of mating type switching is a gene conversion event initiated by the HO
gene. The HO gene is a tightly regulated haploid-specific gene that is only activated in
haploid cells during the G
1
phase of the cell cycle. The protein encoded by the HO gene is
a DNA endonuclease, which physically cleaves DNA, but only at the MAT locus (due to
the DNA sequence specificity of the HO endonuclease).

Once HO cuts the DNA at MAT, exonucleases are attracted to the cut DNA ends and
begin to degrade the DNA on both sides of the cut site. This DNA degradation by
exonucleases eliminates the DNA which encoded the MAT allele; however, the resulting
gap in the DNA is repaired by copying in the genetic information present at either HML
or HMR, filling in a new allele of either the MATa or MAT gene. Thus, the silenced
alleles of MATa and MAT present at HML and HMR serve as a source of genetic
information to repair the HO-induced DNA damage at the active MAT locus.

Figure 7-66 Molecular Biology of the Cell ( Garland Science 2008)
Genomic Control:
Chromosome Decondensation and DNA accessibility
Elaborate packaging of DNA with proteins to form euk. Chromosome adds
a level of complexity.

To initiate transcription RNA pol must interact with DNA and a no. of
specific proteins in promoter region.

Promoter embedded within highly folded and ordered chromosomal
superstructure.

Some degree of chromatin decondensation (unfolding) is a critical event in
euk. gene exp.
Visualization of Chromosome Decondensation
Visual evidence for correlation between chromosome decondensation and
transcription comes from studies on polytene chromosome of Drosophila.

Polytene chr. is a tightly attached pair of homologous chromosomes with a
very large no. of precisely aligned, parallel chromatids. Visible in each of
these chromosomes is a characteristic pattern of dark bands (highly
condensed) and chromatin in a band is uncoiled during transcription.
Transcriptional activity of polytene chromosomes
Transcriptionally active regions
light up under FM
Puffs in Polytene chromosomes
Activation of genes of a given chromosome band causes coiled chromatin
strands to unwind and expand outward resulting in a chromosome puff.
Puffs are regions in which transcriptionally active region has become less
condensed.

The puffing is direct visual manifestation of selective decondensation and
transcription of specific segments of DNA
Year Who Award
1910
Albrecht Kossel (University of
Heidelberg)
Nobel Prize in Physiology or Medicine "in recognition
of the contributions to our knowledge of cell chemistry
made through his work on proteins, including the
nucleic substances"
1933
Thomas Hunt Morgan
(California Institute of
Technology)
Nobel Prize in Physiology or Medicine "for his
discoveries concerning the role played by the
chromosome in heredity"
1962
Francis Crick, James Watson
and Maurice Wilkins (MRC
Laboratory of Molecular
Biology, Harvard University and
London University respectively)
Nobel Prize in Physiology or Medicine "for their
discoveries concerning the molecular structure of
nucleic acids and its significance for information
transfer in living material"
1982
Aaron Klug (MRC Laboratory of
Molecular Biology)
Nobel Prize in Chemistry "for his development of
crystallographic electron microscopy and his
structural elucidation of biologically important nucleic
acid-protein complexes"
1993 Roberts and Sharp
Nobel Prize in Physiology "for their independent
discoveries of split genes"
2006
Roger Kornberg (Stanford
University)
Nobel Prize in Chemistry "for his studies of the
molecular basis of eukaryotic transcription"
Nobel Prizes
The following scientists were recognized for their contributions to chromatin research with Nobel Prizes:
Main control Point of gene regulation: Initiation of transcription
A typical eukaryotic gene with its core promoter and proximal control region
A promoter is a DNA sequence that enables a gene to be transcribed. The
promoter is recognized by RNA polymerase, which then initiates transcription.
In RNA synthesis, promoters are a means to demarcate which genes should be
used for messenger RNA creation and thereby controls which proteins the cell
manufactures.

Figure. Primary and secondary levels of gene regulation.
According to this scheme, 'primary' regulation of genome expression occurs at the level
of transcription initiation, this step determining which genes are expressed in a
particular cell at a particular time and setting the relative rates of expression of those
genes that are switched on.
'Secondary' regulation involves all steps in the gene expression pathway after
transcription initiation, and serves to modulate the amount of protein that is synthesized
or to change the nature of the protein in some way, for example by chemical
modification.
Transcription in eukaryotic cell
More complex. Main differences:

1) Three diff RNA Polymerases
2) Euk. Promoters are more varied. 3 diff. types of promoters for 3 RNA
polymerases. Great variation within each type especially one for protein
coding genes.
3) Many transcription factors are involved in binding of RNA Pol. to DNA.
(Most of them must bind to DNA before Pol. can bind to promoter and
initiate transcription. TFs determine specificity of transcription in
eukaryotes).
Eukaryotic RNA Polymerases
Structurally all three are somewhat similar to each other and also to prokaryotic
RNA pol.

Quite large with multiple polypeptide subunits and MW 500,000
Typical eukaryotic promoters used by RNA Polymerases I, II and III
I
II
III
tRNA gene
5S rRNA gene
Promoters
Pol III is unusual
(compared to Pol II)
requiring no control
sequences upstream of
the gene, instead
normally relying on
internal control
sequences - sequences
within the transcribed
section of the gene
The process of transcription
by Pol I is relatively
unregulated (rRNA for
ribosomes is always needed
in large quantities).
Consequently, transcription
by Pol I is a comparatively
simple process with few
steps requiring regulation.
Transcription Factors
A transcription factor is a protein that binds DNA at a specific promoter or
enhancer region or site, where it regulates transcription. Transcription factors
can be selectively activated or deactivated by other proteins

Transcription factors possess several distinct functional domains:

DNA-binding domain
Transcription regulation domain (in activators called as TAV)

Most TFs fall into categories based on structural motif present in DNA-binding
domain.


There are three classes of transcription factors:

General transcription factors are involved in the formation of a preinitiation
complex. The most common are abbreviated as TFIIA, TFIIB, TFIID, TFIIE,
TFIIF, TFIIH. They are ubiquitous and interact with the core promoter region
surrounding the transcription start site(s) of all class II genes.

Upstream transcription factors are proteins that bind somewhere upstream of the
initiation site to stimulate or repress transcription.

Inducible transcription factors are similar to upstream transcription factors but
require activation or inhibition.

DNase I Sensitivity Studies
Demonstrates correlation between chromatin uncoiling and transcription.
At low conc. DNase I preferentially degrades transcriptionally active DNA in chromatin
DNA in condensed
chromatin
protected from
DNaseI
BRAIN ERYTHROCYTE
Globin gene is inactive in in brain cells: Present intact after nuclease treatment
Globin gene is active in erythrocytes: DNA is vulnerable to nuclease attack.
Globin
Ovalbumin (control)