regulation of gene expression in eukaryotes
Methods in which translation initiation regulates gene exp in eukaryotes
-RNAi - Regulatory proteins bind 5' and 3' UTRs of mRNAs
what are the five methods of controlling gene expression in eukaryotes?
1. DNA Packaging 2. Transcription initiation 3. RNA Processing 4. RNA Stability 5. Translation initiation
RNA stability and gene exp (2 topics)
1. length of poly-a tail 2. RNAi
which region of mRNA is important for RNA interference
3' UTR
describe the process of southern blotting
DNA is transferred to a filter paper/membrane and a probe is synthesied. the probe and the membrane are incubated and autoradiography is udef to see where probe hybridizes to membrane. position indicates size compared to control dna ladder of known size.
DNAse I experiments to test hypothesis.
DNAse 1 is an endonuclease that cuts DNA nonspecifically. It was found that the DNA could only be cut if it was accessible/ in the open/ euchomatin conformation. It was found that: the regions in the dna first cut by dnase 1 were usually upstream of the gene where the txn factors and RNAP bind. The researchers wanted to know the conformation of chromatn at known active/inactive genes globin and ovalbumin. First chromatin was isolated from cells and mixed with different amoundts of DNAse I (after being purified from the proteins in tube). The dna is then cut with a restiction enzyme to release a fragment of a known size. the fragments were then put through gel electrophoresis and southern blotting. If the Dna was in the open conformation, DNAse degrades it, if not, it will be cut by restriction enzymes.
gene xp regulation by histone mofication in arabidopsis What is the FLC gene? What is the FLD gene?
FLC gene - codes for a regulator protein that represses activity of genes important for flowering FLD gene - codes for histone deacetylase when flc gene is acetylated, the chromatin structure is destabilided and RNAP is able to bind and FLC gene encodes for regulator protein that represses flowering.. FLD gene encodes hstone deacetylase and acetyl groups are removed. no transcription of flc occurs and flowering is not suppressed. FLD represses FLC.
example of regulation in txn initiation (galactose metabolism in yeast
GAL4 protein (activator) binds to UASG (enhancer), but only when galactose in present because gal4 is repressed by GAL80 and binding. galactose binds too gAL3 which binds to GAL80 and prevents it from binding to silencer and preventing gal4 from stimulating txn
anaother term for RNAi (RNA interference)
RNA silencing
What transcribes pre-mRNA in transcription initiation process?
RNAP II
repressor protein
Repressor proteins bind to silencers (section on gene) or to certain sequences in regulatory promoter to decrease txn repressors may compete with transcriptional activators for binding sites on the DNA: when a site is occupied by an activator, transcription is activated, but if a repressor occupies that site, there is no activation. Alternatively, a repressor may bind to sites near an activator binding site and prevent the activator from contacting the basal transcription apparatus. A third possible mechanism of repressor action is direct interference with the assembly of the basal transcription apparatus, thereby blocking the initiation of transcription.
alternative splice of tra premRNA in flies
SxI gene encodes the regulation of splicing (but only in females) -Causes splicing of premature stop codon that both males and females contain -Females have functioning Tra protein Males have non-functioning protein
how do response elements allow complex biochemical responses in eukaryotic cells?
a single gene may be activated by several different response elements found in both promoters and enhancers. Multiple response elements allow the same gene to be activated by different stimuli. At the same time, the presence of the same response element in different genes allows a single stimulus to activate multiple genes. In this way, response elements allow complex biochemical responses in eukaryotic cells.
Examples of Trans-acting factors
activator proteins, repressor proteins, insulator-binding proteins
where does histone modification occur
amino acid tail of protein
transcription factors are part of
basal transcription apparatus
activator protein
binds to enhancer (section on gene)or regulatory promoter and increases txn -Job of activator is to bind to enhancer then mediator (part of RNAP/basal transcription apparatus) to influence rate of txn.
examples of cell types and processes impacted by control of gene xp in eukaryotes
cell differentation aka the different cell types and how they are regulated, timing for flower development
stimuli
cellular stresses hormones nutrients development etc
promoters have several different ____-acting elements
cis
transcriptional regulatory proteins can either be
cis acting or trans acting factors
what do trans acting factors bind to in regulation of txn initiation
cis-acting elements
basal transcription apparatus
complex of RNA polymerase, transcription factors, and other proteins that assembles to carry out transcription.
what is proper spicing dependent on?
consensus sequences
what kind of sequence do activator proteins bind to
consensus sequences
where does basal transcription apparatus bind?
core promoter located immediately upstream of a gene
where does dnsa methylation happen?
cpg islands
dna methylation correlates with (acetylation/deacytlation) of histone proteins
deacetylation
dna methylation correlates with (increased/decreased) txn?
decreased
what are consensus sequences important for in splicing?
determining the precise location of the introns and exons.
how does the mix of regulatory elements in promoters vary from gene to gene?
different combinations of elements and different numbers of copies of each element are present in different genes
how can alternative splicing regulate gene expression?
different proteins generated from splicing at different times in development
how does dna packing relate to gene xp?
dna is packed into chromatin (DNA+protein complex). tightly packed areas aka heterochromatin ("closed" conformation) which are less accessible to RNA polymerase are inactive/encode for few genes. loosely packed areas aka euchromatin that are accessible to RNA polymerase, are active/encode for many genes. Scientists hypothesised that there is a correlation between the closed/open conformations and transcription initiation.
does histone modification increase or decrese txn?
either depending on which amino acids are modified.
examples of cis acting elements
enhancers, silencers, insulators
Length of Poly(A) Tail and gene expression in eukaryotes
gene expression depends on amount mRNA and how quickly it is degraded - tail is recognized by Poly(A) Binding Proteins (PABPs) - RNases shorten poly(A) tail over time - When PABP can't bind anymore (10-30 A's long), mRNA is quickly degraded
what often creates ans "epigenetic mark"?
hisone modification/methylation?
insulator
insulator binding proteins bind to insulators (create boundaries for enhancers) insulator is DNA sequence that blocks, or insulates, the effect of an enhancer; must be located between the enhancer and the promoter to have blocking activity; may also limit the spread of changes in chromatin structure.
when dna methylation is low and histones are acytylated chromatin conformation is
loose
relationship between methylation and deacytlation
methylated dna is binding site for histone deacetylases
miRNA to translation inhibition
miRNA binds to RISC and attaches to mRNA at 3' UTR and blocks/inhibits translation of mRNA
which molecules trigger RNA interference? (2) (the "small RNAs" mentioned above)
miRNAs and siRNAs
types of histone modifications
phosphorylation, ubiqinaton, acetylation, metheylation
where are gpg islands common
promoters
how does chromatin remodeling work?
proteins reposition nucleosomes on dna to make the dna more accessible to transcription factors. there is no chemical modification of the dna or histones.
cpg islands
regions of dna rich in repeats of cg
where to the regulatory proteins bind?
regulatory promoter and enhancers
promoters in basal txn apparatus
regulatory promoter, core promoter
histone deacetylation
removal of acetyl groups from histone tails allowing for tighter dna packing
explain coordinated gene exp in eukaryotes
several eukaryotes genes are activated by the same stimulus because they all have the same response element within their promoters while some individual eukaryotic genes have multiple response elements and and are therefore activated by different stimuli
siRNA to transcription ihnibition
siRNA combines with complex called RITS then binds to complementary sequence in DNA or RNA then supresses transcription by attracting enzymes that methylate the tails of histone proteins or methylate the DNA itself
siRNA to mRNA degradation
siRNA from sliced rna binds to RISC and then to 3' UTR on mRNA which leads to clevage of the mRNA and then degradation
mechanisms of inteference by the small rnas (3)
siRNA to mRNA degradation, miRNA to translation inhibition, siRNA to trabscribtion ihnibition
RNA interference is mediated by
small RNAs (21-22 nucleotides long)
enhancers are usually located
some distance from the gene
probe
ssDNA oligonucleotide (fluorescent or radioactive) that is complementary to specific sequence
example of a single gene being regulated by several different response elements regulated expression of metallothionein gene
the metallothionein gene encodes a protein that protects cells from the toxicity of heavy metals by binding to those metals and removing them from cells. the txn apparatus alone is capable of only low rates of transcription. Other response elements found upstream of the metallothionein gene contribute to increasing its rate of transcription. For example, several copies of a metal response element (MRE) lie upstream of the metallothionein gene The presence of heavy metals stimulates the binding of transcriptional activator proteins to the MRE, which elevates the rate of transcription of the metallothionein gene. Because there are multiple copies of the MRE, high rates of transcription areTABLE 17.1 induced by metals. Two enhancers are also located in the region upstream of the metallothionein gene. One of these enhancers contains a response element known as TRE, which stimulates transcription in the presence of a protein called AP1. A third response element, called GRE, is located approximately 250 nucleotides upstream of the metallothionein gene and stimulates transcription in response to certain hormones. This example illustrates a common feature of eukaryotic transcriptional control: a single gene may be activated by several different response elements found in both promoters and enhancers. Multiple response elements allow the same gene to be activated by different stimuli.
when dna methylation is high and histones are deacytylated chromatin conformation is
tight
why are transcriptional regulator proteins needed?
to bring about normal levels of transcription
purpose of southern blotting
to show size of specific dna fragment
general picture of txn initiation (w/ activators) in eukaryotes
transcription activators bind to enhancer region dna and to mediator part of basal transcription apparatus. The enhancer may be far from core promoter which causes the dna to loop out so the activator can bind correctly to both the mediator and the enhancer
Fragile X syndrome and cpg islands
transcription of FMR-1 gene is inhibited - non-disease chromosomes have CGG repeat (29 copies) upstream of FMR-1 gene - disease chromosomes have 200-1300 copies of CGG repeat - coding region of gene is same in normal and diseased individuals
where is regulatory promoter located?
upstream of the core promoter
