Week 6 Flashcards
What is the central question in the regulation of gene expression?
How does a cell or organism with the same initial DNA sequence/genotype exhibit different phenotypes?
How does a cell or organism with identical initial DNA sequence/genotype exhibit different phenotypes?
Different environmental conditions
Different cell types
How does a cell or organism with identical initial DNA sequence/genotype exhibit different phenotypes?
Different environmental conditions
Different cell types
Differences between bacteria and eukaryotes
Compartmented;Uncompartmented
Polycistronic mRNA/monocistronic
Coupled transcription and translation/Uncoupled
mRNA primary transcripts not spliced/spliced and modified
One RNA polymerase/multiple RNA polymerases
Bare DNA/Chromatin
Polycistronic mRNA
A single mRNA encodes multiple polypeptides (in viruses the DNA is expressed as polycistronic in a eukaryote)
RNA polymerase eukaryotes
RNAP I (rRNA)
RNAP II (mRNA)
RNAP III (tRNA)
Regulation at the level of transcription (bacteria)
Is the gene the same structure
Is the gene transcribed
Where is the transcript initiaited/terminated (Polycistronic)
Regulation at the level of the transcript (bacteria)
Stability of the transcript
Regulation at the level of translation (bacteria)
Is the transcript translated
Regulation at the level of the protein (bacteria)
Is the protien active?
Regulation at the level of transcription (eukaryotes)
Is the gene the same structure
is the transcript initiated
where is the transcript initiated
where does the transcript end
how was the transcript spliced
Regulation at the level of the transcript (eukaryote)
How stable?
Regulation at the level of translation (eukaryotes)
Is the trancript translated
Where is the transcript
Is the transcript exported from the nucleus
Regulation at the level of the protein (eukaryote)
is the protein active?
where is the protein?
Determining at what level gene expression is regulated?
Is the transcript present or not?
What is the structure of the transcript?
Is the protein expressed?
Is the transcript present or not?
Transcript detection/accumulation
What is the structure of the transcript
transcript analysis/sequencing
Is the protien expressed
Protein detection/antibody or activity
Transformer gene and female development
In Males: OFF
In Females: ON
Is tra transcribed in females only?
Nope tra transcription occurs in both males and females
Is TRA mRNA translated in females
YES TRA protein is only detected in females
Difference in tra mRNA in males and females
Male transcript has extra RNA sequence information that introduces a stop codon in the mRNA.
In females the splicing of the mRNA results in mRNA that lacks this sequence that is present in the male transcript resulting in no premature truncation.
RNAP Role in Transcription
RNAP binds forming a closed complex.
Regulates the rate at which the gene is transcribed/amount of transcript produced.
conformational change to form a bubble.
DNA elements: promoter
RNA polymerase in bacteria recognize the -35/-10 sequence directly by binding
In eukaryotes the TATA box is 30bp upstream of the start of transcription
DNA elements: regulatory sequence role
Rate of transcription are regulated by the regulatory sequence (positive/negative sequences).
Bacteria regulatory sequences
Activator sequence (positive)
Operator (negative)
Yeast regulatory sequences
upstream activation sequence (UAS)
Operator/silencer
Eukaryote regulatory sequence
Enhancer/Silencer
A major difference between prokaryotic and eukaryotic promoters
A transcription unit with a promoter is generally going to be transcribed in prokaryotes.
Promoter is not sufficient for transcription in eukaryotes
Effects of silencer/operators and enhancers/activaters
P: transcription (pro), no transcription (euk)
O/Sil and P: no transcription (pro), no transcription (euk)
A/enh and P: increased transcription (pro) and transcription (euk)
A/enh, O/sil and P: no transcription (pro) and transcription (euk)
Transcription factors
DNA-binding proteins that recognize specific DNA sequences.
When bound to DNA they affect the rate of initiation of transcription
Transcription factors (2)
DNA binding can be influenced by the binding of small molecules.
When allolactose binds to the Lac repressor it can not bind the operator DNA regulatory sequence.
Lac Operon
Transcribed from one promoter and the transcript encodes three proteins.
Beta galactosidase (LacZ) Permease (LacY) Galactosidase acetyltransferease (LacA)
Promotor and Operator part of a gene
laci and lacZ
two seperate genes
LacZ-
Recessive
No betagalatosidase
Oc
Dominant
Betagalactosidase always present
laci-
recessive
Betagalactosidase
laci sr
dominant
no betagalactosiadase
gf
laci encodes a ______ ______that regulates beta galactosidase. the conclusion comes from laci being _____-_____ to lacZ
Diffusable factor
trans acting
The _______ of lacZ is the sequence recognized by the diffusable factor encoded by laci and is required on the _____ ______ as the lacZ coding region. Operator alleles are ____-_______.
operator
same molecule
cis-acting
Genetic analysis of mutant alleles
The experiments showing trans-acting and cis-acting are distinct.
Trans-acting is the conclusion of an intergenic complementation analysis.
Cis-acting is the conclusion of an intragenic analysis of gf and lf lacZ alleles.
Basic Complementation analysis (cis-trans test); LacZ(lf) and Laci(lf)
trans: different molecules
cis: same molecules
Wild type expression occurs in both cis and trans arrangement of the DNA molecules.
laci encodes for a diffusable factor that can bind to operators in either WT gene
cis-acting does not come from a complementation analysis; refers to __-_______
cis-acting refers to cis-dominance which is the interaction between two mutant alleles in the same locus/gene.
To observe cis-dominance, one allele is a dominant gain of function and the other is a recessive loss of function; they have opposite phenotypes.
regulatory sequences are genetic elements within a gene.
Oc allele is _____ to lacZ (lf) allele when trans.
dominant
Wt allele are dominant when Oc and lacZ (lf) are ____.
cis
When we observe cis-dominance does that mean we are dealing with just the same DNA molecule ie. do the elements have to be just on the same DNA molecule?
NO. The elements could be on the same RNA molecule or the same protein molecule.
How can transcription occurring in a cell?
Take advantage of splicing. Place a block of DNA that encodes the MS2 binding site, bound by an RNA binding protein.
The MS2 binding site is places in the intron of the ftz gene such that when a nascent primary transcript is formed it contains the MS2 binding site. After splicing the MS2 binding site is rapidly degraded. The presence of a MS2 binding protein fused to a red fluorescence protein detects the nascent transcripts.
delay between transcription and translation
13 minute delay
Reporter Genes
Reporter expression is easy to assay (green fluorescence)
Reporter genes can reproduce the expression of a gene.
Use of reporter genes requires that you use an organism where it is possible to reintroduce DNA (transform)
Translational fusion to a reporter gene
fuse the regulatory region of a gene to a reporter gene any protein that is easy to assay
Reporter genes can reproduce the expression of a gene
the reporter gene construct has recapitulated the gene expression pattern.
Reporter genes can reproduce the expression of a gene
the reporter gene construct has recapitulated the gene expression pattern.
Protein that regulates splicing of tra mRNA
Sex lethal protein
SXL found in females but not males
Method of splicing used in the tra transcript
Alternative 3’ splice sites used
SXL binds to a specific sequence in RNA (GUUGUUUUUU)
SXL binds to the intron of tra pre-mRNA in females, pushing U2AF to bind the later female 3’ splice site
SXL is a
negative regulator of splicing
if the male 3’ splice site is removed SXL is not required to block its use.
The transcript is spliced in the female manner in both males (SXL -) and females (SXL +)
A mature mrna of the same structure is present, but in one condition it is translated and in another it is not translated
regulation at the level of translation
Hunchback mRNA and Hunchback protein expresison
there is no protein at the posterior end of the cell but mRNA is present in the entire cell.
_____ is required to suppress HB protein expression
Nanos.
LF in the nanos allele results in a difference in how GB mRNA is translated, we see HB protein present at the posterior end
HB expression in the posterior ______ _______ of the _____.
supresses
development
abdomen
What does nanos recognize in HB mRNA to suppress translation
NRE: nanos regulatory element
Pumilio
binds NRE both at the anterior and posterior end of the embryo
no nanos at the anteiror end results in HB mRNA being translated and we get HB protein
How does nanos supress HB mRNA translation
nanos binds to pumilio and recruits 4EHP that binds the cap forming a ring structure that is unable to be translated by ribosomes.
