Gene Expression and Biotech (complete) Flashcards
where are the two levels where regulation of gene expression can occur
- Transcription
2. Translation
What is the difference between cis-acting and trans-acting regulation
cis-acting regulation is transcriptional regulation based on the DNA sequence alone
trans-acting regulation is transcriptional regulation based on something outside of the DNA sequence
Trans-acting regulation factors are usually _______
proteins which bind to the DNA
do trans anc cis-acting regulation factors usually work together? if so how?
trans-acting regulation factors usually bind to the cis-acting factors
Of these, which are cis-acting
- RNA polymerase
- Helicase
- Transcription factors
- promoter
- gene
- enhancer
- repressor
Promotor
Gene
Enhancer
repressor
Of these, which are trans-acting
- RNA polymerase
- Helicase
- Transcription factors
- promoter
- gene
- enhancer
- repressor
RNA polymerase
Helicase
Transcription factors
Is most regulation of gene expression done at translation or transcription
transcription
Where is gene expression regulation done in prokaryotes
only transcription, no translation
What is an operon
a cluster of genes with related functions, acting as a coordinated unit and controlled by a regulatory sequence
What are the components of an operon
- Structural genes
- Promoter
- Repressor
- Activator
- Operator
What are the structural genes of an operon
the genes that code for proteins
what is the difference between the structural genes of an operon in prokaryotes vs. eukaryotes
the structural genes in eukaryotes only codes for one protein, where as in prokaryotes it can code for multiple proteins
Is operon usually used in association with prokaryotes or eukaryotes
prokaryotes
What is the promoter of an operon
- it is the location where the RNA polymerase will bind
- It has the TATA box
- it increases the efficiency of gene transcription
What is the repressor of an operon
- a gene that codes for an inhibitor protein of the gene
2. it inhibits the transcription of the protein
What is an activator of an operon
- it increase the transcription of a protein
2. a gene that codes for an activator protein of the gene
What is an operator of an operon
the sequence of DNA that binds repressor or activator proteins
How many promoters per gene is there in eukaryotes
one promoter per gene
How many promoters per gene are there in prokaryotes
multiple promoters per gene
Genes can be either:
1. Constantly activated and turned off when you have too much of the protein
2. Constantly inactivated and turned on when more of the protein is needed
True or False? if only one, which is it?
True,
In prokaryote transcription, what is the concensus sequence
- the preferred binding sequence for transctiption factors.
- it is about 35 bases prior to the location where transcription begins
- TTGACA is the sequence
in prokaryote transcription, what is the pribnow box
a portion of the promoter that is about 10 bases prior to transcription start point
TATAAT is the sequence
What are the proteins that are a created and regulated by the LAC operon
They are three catabolic enzymes that break down lactose
Which prokaryotic operon is known as the catabolic operon
the LAC operon
Is the Lac operon normally on, or off
the lac operon is normally OFF
When is the lac operon turned on
when lactose needs to be used for energy
When there is high lactose and allolactose
What is the lac operon like in its normal state
- It is off
2. it has a repressor protein (from the repressor gene)that is bound to its operator
How does the lac operon get turned on
- Lactose enters the scene and binds to the repressor protein that is on the operon, this pulls the repressor protein off and allows transcription to begin
What is an inducer
a substance that binds to a repressor, causing it to be inactive
in the LAC operon what is the inducer
lactose
in prokaryotes what is also known as the Anabolic operon
the TRP operon
What are the two main important prokaryotic operons
LAC operon
TRP operon
Is the TRP operon normally on or off
normally on
What causes the TRP operon to get shut off
high levels of Tryptophan
in the TRP operon, tryptophan will bind to an inactive repressor on the operon, causing it to become active, and stop transcription. What is tryptophan acting as
a corepressor
what is a corepressor
something that binds to a repressor, causing the repressor to be activated
What is the product protein of the TRP operon
proteins that are used in the biosynthesis of tryptophan
how many proteins are coded for in the TRP operon
5
Can prokaryotes do translational regulation
yes, but not very often. sometimes the protein made from an mRNA will bind to that mRNA causing it to become inactive
What is the very first method of transcription regulation in eukaryotes
The DNA being bound to histones, making the genes impossible to access until they are removed from the histones
What enzyme will pull the DNA off of histones, allowing the DNA to unwind from the chromatin structure, allowing the genes to be transcribed.
HAT
Histone Acetyltransferase
What does HAT (histone Acetyltransferase) do to pull the DNA off of histones
it acetylates the histone
What enzyme will allow the histones to rebind DNA, form the chromatin structure, and stop any transcription
HDAC
HIstone Deacetylase
What does HDAC do to allow histones to bind to DNA
it deacetylates the histones
Do Eukaryotes require initiation factors and promoter sequences for transcription
yes
what are the different promoter sequences in eukaryotes used to help RNA polymerase begin transcription
- TATA box (25 Base pairs upstream)
- CAAT box (75 Base pairs upstream)
- GC-rich sequence
do most eukaryotic genes have all three of the promoter sequences?
No, most have the TATA box, and EITHER the CAAT box, or the GC-rich sequence.
in Eukaryotes, do hormones help regulate transcription
yes
Do hormones usually enhance transcription, or inhibit it
they usually enhance transcription, but rarely can inhibit it
How do lipid soluble hormones affect gene transcription
the hormone-receptor complex binds to the DNA and directly enhances transcription by assisting RNA polymerase 2 attachement
how else can hormones and other substances (that aren’t able to cross the cell-membrane into the cell) affect transcription
they bind to surface receptors, then they change the cellular chemistry and that can affect transcription
What are the two main molecules in a cell that have their levels either raised or decreased by activated surface receptors
cAMP
Ca++
What are the steps involved in transcription regulation via cell surface receptors
- Molecule binds to the cell surface receptor
- This causes an increase in cAMP (or Ca++)
- This activates CBP (cAMP binding protein)
- CBP phosphorylates CREB (cAMP response element protein)
- CREB then binds to CRE (cAMP response elements) on the DNA, and
What are 4 types of translational control in eukaryotes
- Alternative splicing
- mRNA editing
- mRNA stability
- RNA interference (RNAi)
What is Alternative splicing
in eukaryotes there are multiple introns and exons on a single gene. introns are cut out and exons are left in the mRNA and lead to the resulting protein. Alternative splicing is the ability in eukaryotes to selectively cut out some of the exons along with the introns. This allows multiple different proteins that can be made from one gene
What is mRNA editing
translational control that only happens in eukaryotes. Usually it is enzymes changing out bases
in mRNA editing what is a C usually converted to, and what enzyme does it?
C is converted to U
Cytidine deaminase
in mRNA editing what is an A usually converted into, and what enzyme does it?
A is converted to I (inosine)
Adenosine deaminase
A ribosome reads an I (inosine) as a ______ when translating mRNA
Guanine.
so in effect A–> I = A –>G
What is the purpose of the shine delgarno sequence
AKA the cozac sequence
it is a sequence on the mRNA that recruits the ribosome to the mRNA
What are the three sites of ribosomes, and what happens at each
A site - where the tRNA/amino acid combo enter the ribosome
P site - the 2nd site the tRNA moves to, where the bond between adjacent amino acids is formed
E site - the last site, from where the tRNA leaves the ribosome
what are the subunits of the ribosome, how many are there?
there are two subunits. a large and a small one
does mRNA eventually “expire” or get too worn out to use?
yes
what does mRNA stability do for mRNA
it increases the life of the mRNA
What is RNAi (RNA interference)
a method of silencing the translation of certain genes by controlling the RNA for those genes
What is the thought as to how RNAi developped
it is thought to have developped as an intracellular “immune system” to defend against viral infection
What is the effect that RNAi has on endogenous genes
it controls their expression. keeps them from making proteins
What does RNAi target
double stranded DNA
double stranded DNA is rare in cells, where do we often find it
in RNA viruses
endogenous mRNA
what is the mechanism by which RNAi degrades mRNA
- Dicer protein recognizes double stranded RNA and cuts them into short segments (small interfering RNA, (siRNA))
- RNA-induced silencing complex (RISC) binds one of these siRNA and uses it as a template to hunt its complimentary strands
- RISC then degrades the complimentary strands that it finds
Do eukaryotes also do post-translational modifications
yes
what are the types of post translational modifications that eukaryotes do
- Protein stability
- Additional functional groups
- Propeptide
What are the two classes of biotechnically-derived products
- Proteins produced from recombinant DNA
2. Monoclonal antibodies
What is a common use of recombinant proteins
they are used as therapeutics (rDNA drugs)
what is a common use of monoclonal antibodies
diagnostics tests (pregnancy tests)
What are the 4 different recombinant DNA technologies
- DNA cloning
- Restriction enzymes
- DNA plasmids (Vectors)
- PCR technique
What are the steps in producing recombinant DNA proteins
- Identify the gene of interest from the genome
- Isolate the gene using restriction endonucleases
- Amplify (copy) the gene using PCR technology
- place the gene in a vector
- insert the plasmid into a host cell (transfection)
- Allow host to express protein
- collect protein
What are restriction endonucleases
enzymes that cut DNA in the middle of a strand
What are the two types of cuts that restriction endonucleases can do
Flush ends (cuts straight through both strands) Sticky ends (cuts strands in an offset manner, leaving a few nucleotides open so they can base pair)
Do restriction enzymes cut at random places, or do they cut at certain sequences
they cut at specific sequences
What is the purpose of using PCR technology
to amplify/copy the gene
What does PCR stand for
polymerase chain reaction
What are the steps of PCR technology (used to amplify the gene)
- add initial DNA, primers, and DNA polymerase
- heat DNA to denature the DNA strands
- cool DNA partially so that the primers pair with the DNA (annealing)
- Heat slightly to activate the DNA polymerase.
- Keep repeating steps 2 through 5
What is a plasmid/Vector
a circular ring of DNA containing a specific gene or a set of genes
what are the three things you need to make a plasmid/vector
- DNA
- restriction enzymes
- DNA ligase
once you have made a plasmid with your gene of interest what do you do with it
insert it into a host organism
What happens once you enter your plasmid into a host cell
the host uses it machinery to make your protein of interest
What is transfection
the insertion of a DNA sequence into an organism
What are the different types of transfection
Chemical methods liposomes electroporation/sonoporation magnetofection optical transfection impalefection Gene gun viral tranduction
what is the most common recomdinant DNA drug
insulin
besides the proteins themselves, what things can be the desired products of recombinant DNA
- enzymes
2. antibodies
