Week 6

Question 1

Central question in the regulation of gene expression

Answer 1

How does a cell or organism with the same initial DNA sequence/genotype exhibit different phenotypes

Question 2

Differential expression is due to

Answer 2

Environmental conditions; different cell types

Question 3

Bacteria

Answer 3

Uncompartmented; no organelles

mRNAs polycistronic (generally)

Coupled transcription and translation

mRNA primary transcripts not spliced (generally)

One RNA polymerase

Bare DNA

Question 4

Eukaryotes

Answer 4

Compartmented; nucleus cytoplasm etc.

mRNAs monocistronic (generally)

Uncoupled transcription and translation

mRNA primary transcripts spliced and modified

Multiple RNA polymerases

Chromatin

Question 5

Polycistronic mRNA

Answer 5

A single mRNA encodes multiple polypeptides (in viruses the DNA is expressed as polycistronic in a eukaryote)

Question 6

Transcription/translation eukaryotes

Answer 6

mRNA must be processed before export from the nucleus and translation

Question 7

Central Dogma

Answer 7

Gene
Transcription
Transcript
Translation
Protein

Question 8

Bacteria: Transcription Regulation Question

Answer 8

Is the gene the same structure?

Is the gene transcribed?

Is the transcript initiated?

Is the transcript terminated?

Question 9

Bacteria: Transcript Regulation Question

Answer 9

How stable?

Question 10

Bacteria: Translation Regulation Question

Answer 10

Is the transcript translated?

Question 11

Bacteria: Protein Regulation Question

Answer 11

Is the protein active

Question 12

Eukaryotes: Transcription Regulation Question

Answer 12

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?

Question 13

Eukaryotes: Transcript Regulation Question

Answer 13

How stable?

Question 14

Eukaryote: Translation Regulation Question

Answer 14

Is the transcript translated?
Where is the transcript?
Is the transcript exported from the nucleus?

Question 15

Eukaryote: Protein Regulation Question

Answer 15

Is the protein active?

Where is the protein?

Question 16

Is the transcript present or not?

Answer 16

Transcript detection/accumulation

Question 17

What is the structure of the transcript?

Answer 17

Transcript analysis/sequencing

Question 18

Is the protein expressed?

Answer 18

Protein detection/antibody activity

Question 19

Transformer gene and female development: transcription

Answer 19

Transcription of the tra gene occurs in both males and females, the transcript is present in both cells.

Question 20

Transformer gene and female development: translation

Answer 20

Is TRA mRNA translated in females only?

YES I detect TRA protein in females

Question 21

Structure of tra transcripts

Answer 21

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.

Question 22

Transcription steps

Answer 22

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

Question 23

Transcription DNA elements: Promoters

Answer 23

Promoter in bacteria: -35/-10, RNAP directly binds

Promoter in eukaryotes: TATA; 30 bp upstream of transcription start

Question 24

Transcription DNA elements: Regulatory Sequence

Answer 24

Rate of transcription are regulated by regulatory sequence

positive and negative regulatory sequences

Question 25

Bacteria: regulatory sequence

Answer 25

``` Activator sequence (positive) Operator (negative) ```

Question 26

Yeast: regulatory sequence

Answer 26

UAS (positive) Operator/Silence (negative)

Question 27

Humans/Mice: regulaory sequence

Answer 27

Enhancer Silencer

Question 28

Differences between eukaryotic and prokaryotic promoters

Answer 28

eukaryotic promoters are not sufficient for transcription in eukaryotes a transcription unit with a promoter is generally going to be transcribed in prokaryotes

Question 28

Differences between eukaryotic and prokaryotic promoters

Answer 28

eukaryotic promoters are not sufficient for transcription in eukaryotes a transcription unit with a promoter is generally going to be transcribed in prokaryotes

Question 29

Promoter only

Answer 29

Transcription in prokaryote No transcription in eukaryotes

Question 30

Silencer/operator and promoter

Answer 30

no transcription in both prokaryotes and eukaryotes

Question 31

activator/enhancer and promoter

Answer 31

transcription in eukaryotes and prokaryotes

Question 32

activator/enhancer, silencer/operator and promoter

Answer 32

no transcription in prokaryotes and eukaryotes

Question 33

Transcription factors

Answer 33

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.

Question 34

Lac Operon

Answer 34

transcribed form one promoter and the transcript encodes three proteins: ``` Beta galactosidase (Z): enzyme Permease (Y): transporter Galatosidase acetyltransferase (A) ``` Polycistronic

Question 35

Lac operon regulatory sequences

Answer 35

P and O are part of the gene regulatory sequence of Lacz seperate laci gene encodes the lac repressor

Question 36

LacZ-/Oc

Answer 36

LacZ: recessive lf, no beta galactosidase in glucose/lactose media Oc: dominant, gf, beta galactosidase is always present in the glucose/lactose media

Question 37

laci-/laci-sr

Answer 37

laci: recessive, betagalactosidase is always present laci-sr: dominant, betagalactosidase is not present

Question 38

Operator constuitive allele

Answer 38

Operator is knocked out such that the lac repressor cannot find the operatory site and bind to prevent transcription

Question 39

Trans-acting

Answer 39

Diffusable factor can bind to DNA and regulate transcription The conclusion of an intergenic complementation analysis

Question 40

Cis-acting

Answer 40

the conclusion of an intragenic analysis of gf and lf of alleles

Question 41

The experiment showing trans-acting

Answer 41

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

Question 42

The experiment shows a trans-acting conclusion

Answer 42

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

Question 43

cis-acting analysis

Answer 43

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.

Question 44

cis-acting steps

Answer 44

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

Question 45

How can we transcription occurring the cell

Answer 45

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

Question 46

Reporter genes

Answer 46

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)

Question 47

Easy to assay+recapitulates gene expression patterns

Answer 47

Fuse the regulatory region of a gene to a reporter gene that is easy to assay in frame fusion; ATG and the regulatory sequence

Question 48

Identify cis-regulatory sequences

Answer 48

mutate the regulatory region and look at how expression of the reporter gene is expressed

Question 49

Genetic analysis of RNA cis-acting elements

Answer 49

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.

Question 50

transformer gene and female development

Answer 50

tra mRNA is longer in males and results in no protein tra mRNA is shorter due to alternative splicing results in active gene product

Question 51

SXL expression

Answer 51

in males there is no SXL in females there is SXL expressed

Question 51

SXL expression

Answer 51

in males there is no SXL in females there is SXL expressed

Question 52

Alternative splicing

Answer 52

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)

Question 53

Translation regulation

Answer 53

A mature mRNA of the same structure is present, but in one condition it is translated and in another it is not translated

Question 54

Regulation of hunchback mRNA and protein expression

Answer 54

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

Question 55

What does Nanos recognize in HB mRNA to suppress translation?

Answer 55

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.