Week 6 Flashcards
Central question in the regulation of gene expression
How does a cell or organism with the same initial DNA sequence/genotype exhibit different phenotypes
Differential expression is due to
Environmental conditions; different cell types
Bacteria
Uncompartmented; no organelles
mRNAs polycistronic (generally)
Coupled transcription and translation
mRNA primary transcripts not spliced (generally)
One RNA polymerase
Bare DNA
Eukaryotes
Compartmented; nucleus cytoplasm etc.
mRNAs monocistronic (generally)
Uncoupled transcription and translation
mRNA primary transcripts spliced and modified
Multiple RNA polymerases
Chromatin
Polycistronic mRNA
A single mRNA encodes multiple polypeptides (in viruses the DNA is expressed as polycistronic in a eukaryote)
Transcription/translation eukaryotes
mRNA must be processed before export from the nucleus and translation
Central Dogma
Gene Transcription Transcript Translation Protein
Bacteria: Transcription Regulation Question
Is the gene the same structure?
Is the gene transcribed?
Is the transcript initiated?
Is the transcript terminated?
Bacteria: Transcript Regulation Question
How stable?
Bacteria: Translation Regulation Question
Is the transcript translated?
Bacteria: Protein Regulation Question
Is the protein active
Eukaryotes: Transcription Regulation Question
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?
Eukaryotes: Transcript Regulation Question
How stable?
Eukaryote: Translation Regulation Question
Is the transcript translated?
Where is the transcript?
Is the transcript exported from the nucleus?
Eukaryote: Protein Regulation Question
Is the protein active?
Where is the protein?
Is the transcript present or not?
Transcript detection/accumulation
What is the structure of the transcript?
Transcript analysis/sequencing
Is the protein expressed?
Protein detection/antibody activity
Transformer gene and female development: transcription
Transcription of the tra gene occurs in both males and females, the transcript is present in both cells.
Transformer gene and female development: translation
Is TRA mRNA translated in females only?
YES I detect TRA protein in females
Structure of tra transcripts
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.
Transcription steps
1-RNAP binds to the DNA forming a closed complex: regulates the rate at which the gene is transcribed/amount of transcript produced
RNAP binds to a promoter in both bacteria and eukaryotes
2-RNAP forms an open complex; melted DNA bubble
3-Elongation; RNAP transcribes the gene
Transcription DNA elements: Promoters
Promoter in bacteria: -35/-10, RNAP directly binds
Promoter in eukaryotes: TATA; 30 bp upstream of transcription start
Transcription DNA elements: Regulatory Sequence
Rate of transcription are regulated by regulatory sequence
positive and negative regulatory sequences
Bacteria: regulatory sequence
Activator sequence (positive) Operator (negative)
Yeast: regulatory sequence
UAS (positive)
Operator/Silence (negative)
Humans/Mice: regulaory sequence
Enhancer
Silencer
Differences between eukaryotic and prokaryotic promoters
eukaryotic promoters are not sufficient for transcription in eukaryotes
a transcription unit with a promoter is generally going to be transcribed in prokaryotes
Differences between eukaryotic and prokaryotic promoters
eukaryotic promoters are not sufficient for transcription in eukaryotes
a transcription unit with a promoter is generally going to be transcribed in prokaryotes
Promoter only
Transcription in prokaryote
No transcription in eukaryotes
Silencer/operator and promoter
no transcription in both prokaryotes and eukaryotes
activator/enhancer and promoter
transcription in eukaryotes and prokaryotes
activator/enhancer, silencer/operator and promoter
no transcription in prokaryotes and eukaryotes
Transcription factors
DNA-binding proteins that recognize specific DNA sequences
When bound to DNA they affect the rate of initiation of transcription
DNA-binding protiens recognize specific DNA sequences by specific amino-acid base pair interactions
DNA binding can be influenced by the binding of small molecules.
Lac Operon
transcribed form one promoter and the transcript encodes three proteins:
Beta galactosidase (Z): enzyme Permease (Y): transporter Galatosidase acetyltransferase (A)
Polycistronic
Lac operon regulatory sequences
P and O are part of the gene regulatory sequence of Lacz
seperate laci gene encodes the lac repressor
LacZ-/Oc
LacZ: recessive lf, no beta galactosidase in glucose/lactose media
Oc: dominant, gf, beta galactosidase is always present in the glucose/lactose media
laci-/laci-sr
laci: recessive, betagalactosidase is always present
laci-sr: dominant, betagalactosidase is not present
Operator constuitive allele
Operator is knocked out such that the lac repressor cannot find the operatory site and bind to prevent transcription
Trans-acting
Diffusable factor can bind to DNA and regulate transcription
The conclusion of an intergenic complementation analysis
Cis-acting
the conclusion of an intragenic analysis of gf and lf of alleles
The experiment showing trans-acting
Basic complementation analysis (cis/trans test)
trans: different DNA molecules; wt lacz and lf laci are placed next to each other; lf lacz and laci
we see normal gene expression
function wt copies are placed in cis to each other function normally
The experiment shows a trans-acting conclusion
The wild type Laci gene encodes a factor which will be a a protein that is able to diffuse and interact with the lacZ gene
cis-acting analysis
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 allele is a recessive loss of function; they have the opposite phenotypes.
cis-acting steps
place wt operator cis to lacz lf
place gf operator cis to wt lacz
oc allele is dominant to lacz allele when trans
Oc can’t have an effect on the WT coding region on another molecule, it can only affect the expression of a gene on the same molecule.
wt allele is dominant when oc and lf lacz are cis to each other; normal expression
How can we transcription occurring the cell
take advantage of splicing. Placed a block of DNA that encodes the MS2 binding sites found in RNA and bound by an RNA binding protein
the ms2 binding site is placed in the intron of the ftz gene such that when the nascent primary transcipt 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 fluorescent protein detects the nascent transcrip
Reporter genes
reporter gene 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)
Easy to assay+recapitulates gene expression patterns
Fuse the regulatory region of a gene to a reporter gene that is easy to assay
in frame fusion; ATG and the regulatory sequence
Identify cis-regulatory sequences
mutate the regulatory region and look at how expression of the reporter gene is expressed
Genetic analysis of RNA cis-acting elements
Introduce a change in DNA, that when transcribed results in the production of an RNA that has a sequence change that may or may not affect the regulation of the expression of the protein from this transcript.
transformer gene and female development
tra mRNA is longer in males and results in no protein
tra mRNA is shorter due to alternative splicing results in active gene product
SXL expression
in males there is no SXL
in females there is SXL expressed
SXL expression
in males there is no SXL
in females there is SXL expressed
Alternative splicing
alternative 3’ splice sites used in the processing of the tra transcripts
earlier 3’ ss is used in males,
sxl protein in females binds to sxl binding site forcing the spliceosome complex to bind to a 3’ downstream, the male stop codon is spliced out and active protein product is produced (tra)
Translation regulation
A mature mRNA of the same structure is present, but in one condition it is translated and in another it is not translated
Regulation of hunchback mRNA and protein expression
hunchback mRNA is present in the whole cell
HB protein is only present in the anterior end
Nanos is present at the posterior end of the cell blocking hunch back expression
HB protein expressed at the posterior end results in no abdomen
What does Nanos recognize in HB mRNA to suppress translation?
Nanos requires NRE in order to suppress translation of the HB mRNA (nanos regulatory element)
pumillio another protein expressed in the entirety of the cell and binds to the NRE, pumillio then binds nanos
Pumilio binds NRE both at the anterior and posterior end of the embryo, no nanos at the anterior end, HB mRNA is translated and we get HB protein.
