Gene Regulation in Eukaryotes
What are the similarities and differences of gene regulation in Eukaryotes, as opposed to prokaryotes?
Similarity: Both use DNA binding proteins (activators/repressors)
Differences:
1) Most Eukaryotic genes have their own promoters, as opposed to one promoter per multiple structural genes in prokaryotes.
2) Chromatin is present in Eukaryotes (condensed DNA)
3) Transcription and translation isn't coupled in Eukaryotes, greater diversity in regulation mechanisms.
White gene
Methylated CpG causes gene repression, what are the two reasons why?
1) Impedes binding of transcription factors to promoter region
2) Methylated DNA recruits other proteins that form heterochromatin structure
Why do prokaryotes need gene regulation?
Prokaryotes need to respond to environmental changes by rapidly altering their biochemistry
In order to save energy, gene expression must be controlled.
Describe a negative inducible operon
Active repressor is made, meaning transcription is always off
Inducer binds to active repressor, causes conformational change, repressor pops off operator and transcription is turned on
__________ mutants results in transcription always being turned on.
Constitutive
What is the trp operon function?
OFF (due to condensed DNA , chromatin)
The wild type fruit fly displays a _______ phenotype
red eye
Study of phenotype traits not caused by DNA mutations but by external/environmental factors that switch genes on and off
Epigenetics
Structure genes that encode for proteins involved in metabolism, biosynthesis, or structural roles in cells.
Regulatory genes whose products inhance or inhibit gene expression, (proteins and RNA)
Describe a negative repressible operon
Inactive repressor is made meaning transcription is normally on.
Corepressor binds to repressor and allows repressor to bind to operator, transcription is turned off.
__________ mutants result in transcription always being off.
Noninducible
There are two levels of control, what are they named?
Level 1 is negative repressible, level 2 is attenuation
What are the three ways in which Eukaryotes regulate gene expression?
1) Occurs at transcription initiation by activators/repressors.
2) Chromatin structure alteration
3) RNA processing
The mutant fruit fly displays a _______ phenotype
white eye
Describe alternative splicing, and how is can regulate gene expression.
Alternative splicing occurs when introns and at least one exon is removed during RNA processing.
Alternative splicing regulates gene expression because pre-mRNA can be spliced into more than one mature RNA, yielding multiple proteins.
DNA binding proteins are regulatory proteins that bind to DNA sequences and affect gene expression.
_______ enhance gene expression.
________ inhibit gene expression.
Activators
Repressors
The three structure genes located on the lac Operon are _______, __________, and __________.
Lac Y, Lac Z, Lac A
______ proteins can affect both the gene on the nucleoid as well as genes on plasmid
Trans acting
Describe gene regulation using negative repressible mechanism first when (-) tryptophan, then with (+) tryptophan
Inactive trpR repressor is made and can't bind to operator sequence, transcription is on.
Tryptophan acts as corepressor by binding to inactive trpR repressor, causing conformational change, allowing trpR to bind to operator and block transcription.
Define heterochromatin
Heterochromatin describes rightly packed DNA where transcription is always off.
The nature of the mutation is due to _________
Define this chromosomal rearangement
inversion
chromosome breaks into two pieces, inverts, and reinserts back into the chromosome.
Degradation has to do with the _________ of mRNA
Why does this affect gene expression?
stability
This affects gene expression by affecting the amount of mRNA available to translate
There are two ways repressors work, one way is _____________ where either the repressor or the RNA polymerase binds. This causes _______ between the molecules. If the repressor binds, transcription is ______.
Another way repressors work is both RNA pol and repressor bind to DNA, repressor serves as _______ which inhibits gene expression.
mutually exclusive, competition, inhibited.
roadblock
_______ gene encodes for permease which transports lactose across the cell membrane
Lac Y
Cis acting proteins can only control the expression of genes on the _____ DNA
same
The second level of control is _________, which requires a ________sequence of DNA that contains an attenuator region.
attenuation
leader
Define Euchromatin
Euchomatin refers to loosely packed DNA where transcription is on.
If white gene is inserted into the ___________, white gene is expressed= red eye phenotype.
euchromatin
Stable mRNA has longer _______, and a prolonged __________ window.
half-life, translation
What are the two mechanisms of activators?
Activators bind to DNA, bend it, and allow RNA polymerase to recognize promoter, bind to it, and transcribe gene.
Activators can interact directly with RNA polymerase, creating a complex that allows RNA polymerase to bind to promoter region in order to transcribe a gene.
Lac Z gene encodes for _________, which breaks down lactose into glucose and galactose
beta-galactosidase
________________ are used to study gene regulation of lac operon.
partial diploid E coli
Attenuation occurs when the concentration of tryptophan is present, but not high enough to activate the _____________.
trpR repressor
Gene expression is affected by chromatin alteration because ________ allows for gene expression and _________ does not.
Euchromatin, Heterochromatin
If white gene is inserted into the __________, white gene is not transcribed= white eye phenotype.
heterochromatin
________ RNA has a shorter half-life, and a ______ translation window.
Unstable, short
Define Operon
A set of adjacent structural genes whose mRNA is synthesized in one piece, one promoter for multiple structural genes.
What are regulatory genes?
Regulatory genes encode for proteins that help regulate cellular activity.
How is a partial diploid e coli constructed?
First infection: infect e coli with f/ plasmid, f' plasmid recombines into nucleoid, and recombines out, now containing host gene lac operon.
Second infection: f' plasmid containg lac operon is inserted into new e coli, lac operon is located on the nucleoid and the f' plasmid.
Define the leader sequence on the trp operon, size, location, and special binding site.
160 nucleotides
In between operator and start codon of trpE gene.
contains 5'-UGGUGG-3' where charged tRNA binds (carries tryptophan amino acid)
What are the three processes that cause DNA to switch from Euchromatin to Heterochromatin, an vice versa?
1) Chromatin remodeling
2) Histone modification
3) Methylation of DNA
There are three ways to convert euchromatin<====>heterochromatin, what are they?
1) Chromatin remodeling
2) Histone modification
3) Methylation of DNA
RNA interference involves what molecules?
siRNA and miRNA
Transcription is normally off, something must happen to _______ transcription.
Induce
Negative Inducible is the first level of gene regulation on the Lac operon and it involves the concentration of what substrate?
lactose
The lacI repressor is _________ acting which means the f' plasmid and nucleoid from the mutant e coli are __________ to each other.
if one produces a wild type LacI, it can act on _______ operators
trans
complementary
both
Define attenuator sequence, size, functions
Attenuator sequence is within the leader sequence, 40 nucleotides long, can act as transcription termination site depending on concentration of the amino acid tryptophan.
RNA processing in another way Eukaryotes regulate gene expression, what are the three main reasons?
1) Alternative Splicing
2) Degradation (stability of mRNA)
3) Translation of mRNA (RNAi interference)
Describe the mechanisms of chromatin remodeling
Chromatin remodeling complex binds to the DNA and causes the nucleosome to slide along the DNA., exposing DNA
Chromatin remodeling complex causes conformational change in DNA, exposing DNA
Depending on what DNA is exposed (activator/repressor site, promoter) activation or repression of gene expression
Describe the molecular mechanism of siRNA interference, enzymes/proteins involved
2) ds siRNA binds to RISC (RNA induced silencing complex), RISC unwinds ds siRNA into ss siRNA
3) single stranded anti sense RNA strand binds to RISC and pairs with mRNA due to base pair complementation
4) RISC cleaves and degrades mRNA which silences gene expression
Transcription is normally on, something must happen to _______ transcription.
repress
Catabolite repression in the second level of lac operon regulation and involves what substrate?
glucose
What is IPTG and why is it used in experiments, as opposed to lactose?
IPTG is a synthetic inducer and is good to use because it's not easily hydrolized by beta-galctosidase, meaning its concentration can be kept constant.
Describe the attenuator control mechanism of the trp operon
Segment 1 and 2 are transcribed and translated ribosome stops at segment 2, segment 3 and 4 are transcribed but not translated, allowing them to form a stem loop structure with multiple U's on 4th segment
Stem loop structure with multiple U's acts as transcription termination site
transcripion of trp operon is prematurely terminated on leader sequence
Describe chromatin remodeling
Chromatin remodeling complex binds to DNA, causes conformational change, repositions nucleosome and exposes DNA.
Either activation/repression of gene occurs, according to what is being exposed, DNA binding protein sites/promoter regions.
What are the types of histone modification?
Methylation and Acetylation
simRNA requires a _____________________, which means it can only regulate one gene.
100% match in nucleotide sequence
Negative vs Positive mechanisms refer to the
presence of repressor vs presence of activator
Explain the mechanism of negative inducible (-) lactose (-) glucose
LacI regulatory gene produces an active LacI repressor that binds to the operator sequence of DNA, inhibiting RNA polymerase from transcribing the structure genes.
(saves energy)
Explain mechanism
No.
Because charged tRNA is low, ribosome stall at -UGGUGG- site on segment one, segment 2, 3 and 4 are all transcribed but not translated, mRNA form stem loop between 2 & 3, but no multiple U's
Stem loop canno act as termination site, transcription continues
trp operon is transcribed
What are two modifications of histones and where on the histone do these modifications occur?
Methylation: add/remove methyl group
Acetylation: add/remove acetyl group
occurs on positively charged histone tails.
What part of the histone is modified?
The positively charged histone tails
1) miRNA DNA is transcribed into pri-miRNA
2) Pri-miRNA is processed into pre-miRNA
3) Dicer enzyme converts pre-miRNA into mature ds miRNA
4) RISC binds to ds miRNA and unwinds it into ss miRNA
5) ss miRNA + RISC= complex that binds to mRNA (multiple binding sites)
6) RISC inhibits translation and degrades mRNA which silences gene expression.
Stimulation of protein synthesis is
Induction
Explain the mechanism of negative inducible (+) lactose (-) glucose
The presence of lactose induces transcription because lactose acts as an inducer, by binding to the LacI repressor, causing a conformational change, removing LacI from the operator, and allowing RNA polymerase to proceed.
(produces energy)
RNA polymerase with sigma 70 transcribes what kind of genes?
Housekeeping genes, proteins made for basic cellular function
Describe methylation of histones and the outcomes
Histone methyltransferase (HMT) is responsible for adding methyl groups
Histome demethylase (HDM) is responsible for removing methyl groups
Methylation either activates or represses gene expression.
Describe methylation of histones, enzymes involved, outcomes.
Histone methyltransferase adds methyl group
Histone demethylase removes methyl group
Trimethylation of histone 3 core on lysine 4 = activation
Dimethylation of histone 3 core on lysine 9= repression
What is an inducer? An example of an Inducer in the Lac Operon?
Lactose is the inducer in the Lac Operon example
What is the overall purpose of transcribing the Lac operon?
The Lac operon encodes for proteins that break lactose down into glucose, the preferred energy source.
RNA polymerase with sigma 24 transcribes what genes?
Genes associated in producing sigma 32, heat shock sigma factor
Describe acetylation and the outcome
Deacetyltransferase removes acetyl group
Outcome: activates gene transcription because chromatin is relaxed/open structure with added acetyl groups.
Describe histone acetylation, enzymes involved, outcomes
Histone acetyltransferase adds acetyl group
Deacetyltransferase removes acetyl group
Activates transcription
___________ inhibits protein synthesis
Repression
What is the enzyme and process involved in repression?
Catabolite repression
Adenylate cyclase is responsible for turning ATP into CAMP
CAMP binds to CAP protein, which binds to the CAP binding site on the DNA, aiding RNA polymerase to bind to the promoter.
High concentration of glucose inactivates adenylate cyclase, inhibiting RNA polymerase from binding to the promoter = no transcription.
RNA polymerase with sigma factor 32 transcribes what genes?
Heat shock response genes
DNA methylation involves two enzymes ________ which adds mehtyl group, and _________ that removes methyl group
_________, located near transcription start site/promoter, is the region of DNA that methyl groups are added or removed.
methyltransferase
demethylase
CpG island
Describe methylation of DNA, enzymes involved, outcomes
methyltransferase adds methyl group = repression
demethylase removes methyl group = activation
What is a corepressor? What is an example of a corepressor in Trp Operon?
Corepressors are molecules that bind to repressors and activate them
Tryptophan is the corepressor in the Trp Operon example.
What happens when the concentration of glucose is low?
Active adenylate cyclase, active RNA polymerase, transcription is on.
What do heat shock genes encode for?
Proteins responsible for refolding nonfunctional proteins into functional proteins in the cell.
CpG - methyl group results in________
repression of transcription, gene expression is off because it impedes the binding of transcription factors to promoter region and methylated DNA recruits other proteins that form heterochromatin (tightly packed DNA)
the activation of transcription, gene expression is on
a region on DNA, located near transcription start site/promoter that contains high frequency of CpG dinucleotides.
If the glucose concentration is low, what also must be present in order for the lac operon to be transcribed?
lactose!
Describe the heat shock response mechanism.
Normal temp: sigma 32 is made but degraded by protease
high temp: sigma factor doesn't degrade, produces heat shock proteins so cell can survive environmental change.