TEST 3 MATERIAL

Question 1

How was the role of DNA discovered?

Answer 1

A type of mold was mutated by chance so that it would not produce an essential amino acid. This was observed by feeding it all 20 amino acids, seeing that it would survive, then only giving the essential ones, and observing that it would die. Then, the amino acid that the mold could not produce was given to it with the essential amino acids to see which gene was mutated.

Question 2

What direction is RNA synthesized relative to DNA?

Answer 2

3’ to 5’, which is down stream since RNA is synthesized from 5’ to 3’, the corresponding peice of DNA is in the opposite direction

Question 3

What are the 2 types of polymerases used in transcription found in bacteria, and what are their functions?

Answer 3

Core enzyme: elongates RNA

Holoenzyme: initiates transcription

Question 4

What is a sigma factor?

Answer 4

it is an enzyme that binds the RNA polymerase to a promoter on the DNA sequence

Question 5

What are the 3 steps of transcription? describe them

Answer 5

Initiation: the holoenzyme binds to the promoter (2 sites at -10 and -35 bp) and unwids 10 bp.

Elongation: The polymerase elongates the strand, the new base pairs interact with the template strand briefly to stabilize RNA and poly.

Termination: terminator sequence stops RNA poly, it forms a hairpin that blocks it.

Question 6

Do RNA polymerases need primers?

Answer 6

NO!

Question 7

How is gene expression regulated in prokaryotes?

Answer 7

Transcription factors and activators help RNA poly bind to the promoter in order to express a certain gene, while repressors will block the RNA poly from transcribing. These proteins bind to operons which are regions of DNA controlled by a single promoter. This means that a gene can be positively regulated or negatively regulated.

Question 8

What is a polycistronic RNA and what is advantageous about having polycistronic RNA? Why can’t this occur in eukaryotes?

Answer 8

it is a peice of RNA that includes the code for more than one gene, and can code for more than one protein. I is advantageous because it is more efficient in translating RNA to proteins.

Because the ribosomes are located outside the nucleus in eukaryotes, translation cannot begin immeadatly after transcription. Thus making polycistronic RNA an less effective way of transcribing RNA

Question 9

What is induction and repression and how is it different from positive and negative control?

Answer 9

induction is when the transcription of a gene is positively influenced by the pressence of a chemical, and repression is when the transcription of a gene is negatively influenced by the presence of a chemical.

positive and negative control deal with how repressors influence translation, rather than other nutrients or chemicals

Question 10

What types of RNA polymerases are found in eukaryotes and what are their roles?

Answer 10

Polymerase I, transcribes rRNA
Polymerase II, transcribes mRNA
Polymerase III, transcribes tRNA

Question 11

How does initiation take place in eukaryotes?

Answer 11

transcription factors bind to the DNA and allow DNA poly II to bind to the promoter, containing the TATA box.

Question 12

Why is post transcriptional modification of RNA necessary in eukaryotes?

Answer 12

because RNA is unstable, and it must exit the nucleus and avoid being degraded by other enzymes. In prokaryotes, translation begins as soon a strand of RNA is available.

Question 13

What components are added to mRNA before it exits the nucleus?

Answer 13

A poly-A tail and a 5’ methyl-G cap are added to the ends of the mRNA. These changes prevent mRNA from degrading

Question 14

explain how mRNA can be edited in eukaryotes

Answer 14

a single gene contains introns and extrons. introns are portions of DNA that are spliced out of the mRNA by the spliceosome inside the nucleus. different exons may also be spliced out of the mature mRNA.

Question 15

How does the spliceosome cut out introns of mRNA?

Answer 15

they bind to each end of the mRNA then bring them together making a loop. they then splice both ends, making a lasoo

Question 16

Which mRNA codons start and stop translation?

Answer 16

Start: AUG
Stop: UAG, UGA, UAA

Question 17

What are the different types of mutations and how do they affect protein translation?

Answer 17

Silent: changes a base pair, but still codes for the same amino acid, protein is unaffected

Missense: changes a base pair, changing which amino acid is coded for. This changes the protein in one amino acid

Non-sense: a base pair is changed, which creates a stop codon. This causes translation to stop prematurely, making a short protein

Frameshift: one or two base pairs are added or removed. This completely changes the reading frame and causes extensive missense and potentially non-sense mutations.

Question 18

Explain how mRNA is turned into a protein by a ribosome

Answer 18

an amino acid is added to the tRNA by aminoacyl-tRNA, then the tRNA uses a an anti-codon loop to bind to the correct sequence in the mRNA in the A site of the ribosome. The tRNA is then passed to the P site of the ribosome. a peptide bond then forms between a new tRNA arriving in the A site, and the previous tRNA’s polypeptide. The tRNA without any amino acid is passed to the E site where it leaves the ribosome.

Question 19

Why are some codon/anti codon pairings imperfect?

Answer 19

It is because there are more codons than there are tRNA molecules. Thus, tRNA can use i on the last codon to bind to the codon. i does not bind perfectly to other bases. This is called a wobble base pairing.

Question 20

How is translation terminated?

Answer 20

the release factor binds to a stop codon in the A site of the ribosome, pushing the tRNA that is attached to the poly peptide to exit the ribosome.

Question 21

What is an STR and why is it important in PCR reactions?

Answer 21

An STR, short tandem repeat, is a sequence of repeating codons found in junk DNA. the number of repeats varies from individual to individual, so these STRs can be used to differentiate between people

Question 22

Explain the 3 steps, the objective, and name the reactants ina PCR reaction

Answer 22

Objective: to amplify a targetted sequence of DNA

Denaturation: DNA is heated to 95 C to break bonds between both strands
Annealing: The DNA is cooled to allow primers to stick to the targetted region of DNA.
Extension: TAQ polymerase extends the DNA sequence.

components: TAQ poly, primers, free nucleotides, template DNA.

Question 23

What is electrophophoresis? How does it work?

Answer 23

electrophoresis is the process in which DNA is put into an agar gel with a current through such a gel. DNA is negatively charged, so it will go towards +. the bigger fragments will be slower, and the smaller ones will be faster. thus it differentiates DNA based on length.

Question 24

What is molecular cloning?

Answer 24

molecular cloning is the process in which the PCR product is inserted and expressed into another organism.

Question 25

Why can't the PCR product be directly inserted into the organism? How do we solve this issue?

Answer 25

restriction enzymes will degrade it, to solve this problem, the DNA is put into a plasmid, a circular cloning vector which cannot be degraded by restriction enzymes.

Question 26

What are restriction enzymes, and where in the DNA sequence do they cut?

Answer 26

They are enzymes that cut DNA at restriction sites, which are sites where the complementary DNA strands form a palindrome, on one side of the cut the DNA is the same but backwards on the other side of the cut on the complement strand.

Question 27

What are the components of plasmids, and how do they work?

Answer 27

Origin of replication: allows the plasmid to replicate with the bacteria Multiple cloning site: Allows us to insert the gene we want into the plasmid Selective marker: Antibiotic resistance, this is of great use to the bacteria that take in the plasmid, as they will be exposed to an antibiotic Inducible promoter: allows us to express the gene found on the plasmid, usually the lactose promoter is used.

Question 28

What processes help bacteria take up plasmids in transformation?

Answer 28

1: competent cell treatment 2: heat shock 3: electroporation

Question 29

Why is it important that the antibiotic resistance gene, the selective marker, to be in the plasmid?

Answer 29

Bacteria that undergo transformation will not keep a plasmid if it is not useful for their survival, it costs a lot more energy to replicate and to express the genes on the plasmid.

Question 30

What is an RFLP and how is it used in a southern blot or in DNA finger printing?

Answer 30

An RFLP, or restriction fragment length polymorphisms are segments of DNA that differ from person to person where restriction enzymes can cut. A polymorphism is a version of a mutated allele. Because different people have different RFLPs, when exposed to certain restriction enzymes, different people's genomes will be cut at different restriction sites, allowing for DNA fingerprinting. In a southern blot, a radioactive probe will bind to a complementary DNA strand to check for specific RFLPs, to see if a person has a specific allele.

Question 31

What are ddNTP's and how are they used to sequence a genome?

Answer 31

A ddNTP is a nucleobase that does not have an OH group on the pentose sugar. This means that they terminate the synthesis of DNA, because there is no 3' end to bind the next nucleotide. Putting these nucleotides with normal nucleotides in 4 seperate PCR reactions (1 for each base pair) will result in each DNA strand of the PCR product to have a random length. The length of each DNA strand in each sample is determined by putting the 4 samples through electrophoresis and seeing which strands are the smallest. Using this information, becasue each ddNTP reacted seperately, we can tell which base pairs are in which order.

Question 32

What is the function of reverse transcriptase and why is it useful?

Answer 32

Reverse transcriptase can transcribe RNA into cDNA (complement DNA). This allows us to store the cDNA, as it is much more stable than cDNA, use the cDNA to be inserted into a plasmid, and it allows us to know which genes are activated in certain cells to better understand the genome.

Question 33

What is CRISPR and how does it work?

Answer 33

CRISPR is the combination of a CAS9 protein, who can cut DNA, and a guide RNA. The guide RNA binds to the site which is homologous to itself, and the CAS9 cuts the DNA in two. The cell then activates a repair pathway in which it takes the homologous DNA from the other chromosome and uses it as a template to fix the damage. However, the cell can be tricked into repairing the damage with a different piece of DNA that has homologous portions, but other genes as well. This allows us to modify the genome of a cell.