u3 aos 1

Question 1

protein

Answer 1

large complex molecule made out of amino acids
- polypeptide chain

Question 2

what is a protein made of

Answer 2

amino group, r group, carboxyl group and a hydrogen

Question 3

primary structure

Answer 3

a single strand of animo acids

Question 4

secondary structure and what shapes are they

Answer 4

the single strand of amino acids with interactions between them
- alpha helixes (spirals)
- beta pleated sheets
- random loops

Question 5

tertiary structure

Answer 5

irregular folding of the secondary structures. makes a 3D shape

Question 6

quaternary

Answer 6

2 or more polypeptide chains interacting to make a protein

Question 7

what does an enzyme do

Answer 7

speeds up chemical reactions

Question 8

where do things bind to on enzymes

Answer 8

active site

Question 9

what is an active site

Answer 9

active sites are a groove on the enzyme where substrates are captured and undergo reactions. active sites are complementary (specific) to s specific shape of a substrate.

Question 10

what happens to the substrate on an active site

Answer 10

broken apart, or combined to form products

Question 11

competitive inhibitors

Answer 11

bind to the active site to prevent the substrate from binding

Question 12

non competitive inhibitors

Answer 12

bind to the enzyme to change the shape of the active site

Question 13

what does temperature do to enzymes
hot -> cold
cold

Answer 13

too hot of a temperature causes the enzyme to denature (unfolding of polypeptide chains)
and cooling it down after its too hot causes the active site to change shape

too cold means that theres less movement of molecules, meaning theres less likely collisions

Question 14

what does pH do to enzymes

Answer 14

too high/low of a pH changes the protein shape

Question 15

what are nucleaic acics

Answer 15

made of subunits of nucleotides that form RNA and DNA

Question 16

how to know if its 5’ or 3’

Answer 16

look at the points on the carbon and which ones are bonded

Question 17

differences between RNA and DNA

Answer 17

DNA double stranded
DNA is found in the nucleus in eukaryotes
RNA has one less oxygen than DNA

Question 18

process of transcription

Answer 18

1. DNA polymerase binds to the promoter region of the target gene. It unwinds the DNA
2. RNA polymerase reads the
   exposed DNA template strand in the 3’ to 5’ direction to create a complementary 5’ -3’ RNA strand
3. once it reaches a termination sequence, the polymerase will detach the RNA strand

Question 19

post transcription modification (3 things)

Answer 19

• addition of a polyadanine tail added to the 3’ end to stabilise the mRNA
• addition of methyl guanosine to the 5’ end to protect from enzyme attacks
• removal of introns and splicing of the extrons back together

Question 20

process of translation

Answer 20

1. the mRNA strand goes through a ribosome in the 5’ to 3’ direction, where the ribosome will read the codons, initiating polypeptide synthesis when it encounters a start codon.
2. as the ribosome reads the codons in the mRNA, tRNA with complementary anticodons to the mRNA codons will bring the correct amino acids to the ribosome
3. the ribosome joins the amino acids together to make a polypeptide chain until a stop codon is read. This terminates translation

Question 21

how does the protein get out of the cell

Answer 21

exocytosis
1. Polypeptide is translated and folded at the Rough Endoplasmic Reticulum
2. Protein is transported by a transport vesicle to Golgi Apparatus for final modifications and a secretory vesicle is formed around in preparation for export
3. Secretory vesicle moves to plasma membrane and fuses via exocytosis, releasing the protein into the extracellular environment

Question 22

what is a structural gene

Answer 22

genes that code for proteins for a specificl cells need

Question 23

what is a regulator gene

Answer 23

genes that help switch on/off structural genes

Question 24

why are regulator genes needed

Answer 24

can result in the uncontrolled expression of an unwanted gene

Question 25

what is an operon

Answer 25

a group of prokaryotic genes with a related function that are grouped and transcribe together
- only has one promoter region

Question 26

what is an operon made of

Answer 26

structural genes
promoter - where RNA polymerase binds to
operator- where the repressor protein binds to inhibit transcription

Question 27

regulatory gene

Answer 27

produces the repressor protein which will inhibit the transcription of an operon

Question 28

how does an active repressor work

Answer 28

attaches to operator region to block RNA polymerase from transcribing the following structural genes

Question 29

trp operonand what type of operon is it

Answer 29

made to synthesise the animo acid tryptophan. is a repressible operon which means that it is always on unless something turns it off

Question 30

what is an inactive repressor

Answer 30

a repressor gene that has to have something bind to it to become active (the right shape to bind to the operator region)

Question 31

how to stop trp operon (repressor)

Answer 31

1. once enough tryptophan has been produced, the tryptophan will bind to the repressor protein
2. the repressor protein is now the right shape to fir into the operator region
3. the bound repressor blocks the RNA polymerase from moving along the operon to continue transcription

Question 32

attenuation

Answer 32

back up way to stop the transcription of tryptophan

Question 33

how does attenuation work

Answer 33

the leader sequence is transcribed, it forms an mRNa strand that binds together to become a hair pin which stops the transcription of the trp operon

Question 34

how does attenuation work

Answer 34

1. in prokaryotes, transcription and translation occur at the same time
2. when there are high levels of tryptophan, there RNA polymerase moves along the operon quickly. This means that the leader sequence is also read quickly.
3. this causes the mRNA in the leader region, specifically domain 3 and 4, to create a termination hairpin loop
4. this causes the ribosome to be unable to translate anymore, causing both the ribosome and the polymerase to fall off, stoping the production of more tryptophan.

Question 35

what is a restriction enzyme

Answer 35

used to cut up double stranded DNA into smaller segments to be modified

Question 36

what are restriction enzymes for

Answer 36

cut out viral DNA before it gets transcribed by the bacteria

Question 37

what are plasmids

Answer 37

circular DNA in a bacteria that carries genes that aren’t necessary for the bacteria to live, but give survival advantage

Question 38

what are the 2 types of fragment ends

Answer 38

sticky ends - overhanging exposed sequences with no base pairs. can only recombine with complementary ends

blunt ends - no overhanging area

Question 39

why are sticky ends prefered

Answer 39

they allow for only DNA with the complementary bases to recombine in the correct orientation, while blunt ends do not prevent the DNA from connecting the wrong way around

Question 40

how are ends rejoined

Answer 40

DNA ligase create bonds between the phosphate sugar backbone to join the fragments together

Question 41

how is a recombinant DNA produced

Answer 41

1. the DNA plasmid is split by the restriction enzyme on the recognition site to create an opening
2. the same restriction enzyme would be used to cut the gene of interest
3. the gene of interest is inserted into the plasmid at the complementary ends
4. DNA ligase comes to reconnect the 2 DNA strands by bonding the phosphodiester bonds between ENA fragments

Question 42

how does a transformed plasmid get added into bacterial cells

Answer 42

add calcium ions that bind to the surface of the membrane (membrane is negative) and then increase the temperature, and decrease very quickly to create adhesion zones for the plasmid to enter

Question 43

whatcan you do to tell if a plasmid has become recombinant (add ____)

Answer 43

adding extra genes that will help select out bacteria that has been transformed

Question 44

what are antibiotic resistance genes used for in recombinant plasmids

Answer 44

can be added to plasmids to use when testing which bacteria have taken the plasmid.

when testing on plates, have a control (nothing on the plate) and which bacteria grows

another plate with the antibiotic, take some colonies from the first plate and grow it one the second. the ones that do grow are ones with the plasmid as they have the antibiotic resistance gene

Question 45

what is a reporter gene + example with fluorescence gene

Answer 45

a gene that has been added to the plasmid before the gene of interest.
if cut by the gene of interest, it shows that the gene has been inserted into the plasmid.

example: if a fluorescence gene was the reporter gene, the gene of interest would cut the fluorescence gene. colonies that are not bright green are ones that have taken up the gene of interest

Question 46

what is CRISPR

Answer 46

clustered regularly interspaced short palindromic repeats
bacterial immune system that recognises and cuts DNA of invading virus

Question 47

what is the CRISPR array

Answer 47

stores DNA from previous virus invasion to recognised and defend against those viruses

it is a series of repeats and spacers (spacers being the virus DNA) which are transcribed to be inserted into Cas9 proteins

Question 48

how does DNA get added to CRISPR array

Answer 48

other Cas protein have that job

Question 49

what are cas genes

Answer 49

genes that code for the protein cas to be made

Question 50

how are cas 9 proteins made

Answer 50

1. cas protein is made
2. transcription of CRISPR array reads all the different virus DNA and creates complementary RNA strands
3. the RNA strands get cleaved into smaller pieces called crRNA
4. the crRNA joins with tracrRNA which stabilises the crRNA and makes it fit properly into the Cas protein. it is now called gRNA
5. the gRNA is joined to a cas protein (becoming a Cas 9 Complex) , which will then use the gRNA to bind to the virus DNA and cut where it needs to.

Question 51

why is gRNA used

Answer 51

it is complementary to teh bases of the virus, acting as an active site for the enzyme. once it recognises the DNA, it will break the phosphodiester bonds within the virus and

Question 52

what are PAM sequences

Answer 52

sequences that the Cas 9 uses to bind to DNA and then check to see if there’s viral DNA to cut

Question 53

why do cas 9 proteins not target the CRISPR array

Answer 53

Cas 9 proteins look for PAM sequences, which are not found in the CRISPT array, as they are only made of spacers and repeats.

Question 54

GMO techniques (5)

Answer 54

gene therapy - removing faulty genes and replacing it with the correct codon

gene editing- changing DNA code without removing of the entire gene (CRISPR)

cloning- creating a genetically identical cop of an oranism

transgenic modification - inserting a gene from a difference species into the DNA of an organism

gene silencing - removing or silencing a faulty gene

Question 55

pcr

Answer 55

Denaturation at 94˚C
Heating everything up to 94˚C causes the DNA strand to separate as hydrogen bonds between nucleotides break

Annealing at 55˚C
Two primers are needed that attaches to the beginning or end of our target DNA region 🡪 if they attach in the middle of the target region then we would not have the entire sequence amplified

Extension at 72˚C
The temperature of the PCR is raised to 72˚C which within the optimum temperature range for Taq polymerase to attach and build the sequence
Attaches to the primers on the template strand
Works in 5’ to 3’ direction, adding free nucleotides to the exposed strand to make a complementary sequence

Question 56

how is human insulin made

Answer 56

1. insulin needs to lack introns (using cDNA or synthetic DNA) + needs to have stick ends (EcoR1 and BamH1)
   - if that the ecoli has taken in the recombinant genes, then then it will be ampicillin resistant, but not tetracycline
2. test one plate with ampicilin
3. test one plate with tetracycline
4. reuse EcoR1 and introduce lacZ gene
5. ecoli takes in the recombinant plasmid.

the methionine added to the sticky end of the insulin A gene can be broken down by cyanogen bromine to seperate the blue thing, from the insulin

Isolate genes for two different insulin polypeptides (1) and ligate/use DNA ligase to insert into two different plasmids into the β -galactosidase gene (1) and transform into two separate bacteria (1). Plate on agar containing X gal and ampicillin – those transformed bacteria turn blue (1). Once the genes are expressed and the fusion proteins are produced by each bacteria, these fusion proteins are then purified (1), and the insulin polypeptides are removed and then combined together to produce functional insulin (1).