Section 2: Basic Mechanisms of Gene Expression (Cards for Lectures 9-11)

Question 1

Simple Sequence DNA

Answer 1

• Repetitive sequences of DNA
• Make up about 6% of the human genome
• Two different types: Satellite DNA, which is 14-500 bp repeats in tandem for a total of 20-1000kb, and Microsatellites, which are 1-13 bp repeats in tandem, up to 150 bp
• Mose repeats are non-functional
• Most repeats are in a fixed position
• People can have a different number of repeats due to
• • Replication error
• • Backwards slippage: a loop of one of the repeats, causing an extra repeat to appear after the strand with the loop occurs. This occurs b/c the cell is copying so many of the same repeats
• • Number of repeats can change due to unequal crossing over in meiosis, in which one chromosome gets x more repeats than the original chromosome, and the other gets x less repeats than the original chromosome, so the two new chromosomes vary by 2x numbers of sequences
• PCR of these regions can be used for identification in DNA fingerprinting, and paternity determination

Question 2

DNA Fingerprinting

Answer 2

• Looks at this repeat difference in certain sequences in different people to identify someone from a piece of DNA found somewhere
• PCR is carried out on each suspect’s DNA, and the same primers are able to be used on each person because the location of these repeats is the same in the genome for everyone, even though people differ in the different numbers of repeats
• This is typically done for multiple simple sequence DNA locations, since some people may have the same number of repeats for one simple sequence, but the chances of them having the same for all sequences tested is unlikely
• The PCR product, which will be just the sequences, is then run on a gel, with the location of the band corresponding to the number of repeats in that person’s DNA
• The results of each person is then compared to the DNA found at the crime scene to identify who is the criminal

Question 3

Paternity Determination

Answer 3

• Can do PCR on various simple sequence DNAs to identify father
• The child has two chromosomes: 1 from dad and one from mom. This means that for each repeat sequence, the child will have two copies, and most likely two different numbers of repeats, with one matching up with dad and one matching up with mom.
• A couple different sequences can be selected, PCR can be run, and they can run on the gel.
• Half of the child’s bands should match up with their mom, and the other half should match up with their dad, so by running multiple potential father’s PCR reactions against the child and the mother, it can be determined who is the father by matching up the bands of the child to the father that don’t match up with the mother
• Crossing over in the middle of these sequences complicates things, however, and the child can result in bands that don’t match up with the mother or father.

Question 4

What was the Reverse Transcriptase found in?

Answer 4

• Found in a virus called a “retrovirus’ that has a single stranded RNA as its genetic information
• The reverse transcriptase was able to replicate sRNA and create a new strand of DNA
• Reverse transcriptase is an RNA dependent DNA polymerase
• Called “RNA dependent” b/c it using RNA as its template
• Carries out reverse transcriptions, which is going from RNA to DNA

Question 5

DNA dependent polymerase?

Answer 5

• Something that is DNA dependent uses DNA as its template

- Most DNA polymerases are DNA dependent

Question 6

Process of Reverse Transcription in Retrovirus

Answer 6

• The reverse transcriptase first transcribes ssRNA into cDNA through RNA dependent DNA polymerase action
• Reverse transcriptase degrades ssRNA so we are left with ss-cDNA through RNA nuclease action
• Reverse transcriptase then transcribes complementary strand to form dsDNA through DNA dependent DNA polymerase action
• Integrase then takes the newly synthesized dsDNA and injects it into the human genome
• The dsDNA codes for the reverse transcriptase, the integrase, and the proteus(protease?) virus
• When the time is right, this dsDNA will be transcribed back into mRNA to encode for these proteins so the virus can continue to infect cells

Question 7

Reverse Transcriptase

Answer 7

• Originally found in retroviruses, it has three main enzymatic activities
• It can carry out RNA dependent DNA polymerase action, which is the process of making cDNA from ssRNA, which is what this enzyme is most known for
• It can degrade RNA via RNA nuclease activity
• It can carry out DNA replication/transcription to turn the cDNA into ssDNA via DNA dependent DNA polymerase activity
• It carries out the first three steps of reverse transcription through these activities
• Requires a primer to be able to do reverse transcription and normal replication?

Question 8

Integrase

Answer 8

• An enzyme that is made by retroviruses that injects the ssDNA made from the virus into the human genome

Question 9

HIV

Answer 9

• An example of a retrovirus
• Once the retrovirus inserts its dsDNA into your DNA, you can’t get it out; this is true of all retroviruses
• HIV drugs are typically a “cocktail” of reverse transcriptase inhibitors, integrase inhibitors, and protease inhibitors

Question 10

Reverse Transcription in Eukaryotes

Answer 10

• Mobile elements in eukaryotes can undergo reverse transcription

Question 11

Mobile elements

Answer 11

• Elements of DNA found in eukaryotes genomes that are not “static,” meaning they can move locations in the genome and move between chromosomes
• They are able to undergo reverse transcription to be able to change their location
• About 44% of the human genome contains these mobile elements
• Two main types of mobile elements are DNA transposons and Retrotransposons

Question 12

DNA Transposons and Transposition

Answer 12

• Mobile elements of DNA that are able to move location in the genome via DNA transposition
• DNA transposition follow a “Cut and Paste” mechanism by cutting the transposon out of the donor DNA and pasting it into the target DNA

DNA Transposition:

• The transposon is first transcribed into RNA and then translated into transposase
• The transposase then cuts the DNA transposon out of the donor DNA (via restriction enzyme activity?) and then integrates it into the target region of DNA
• I think the host cell’s machinery then ligates it back together
• There are a LOT of DNA transposons in our genome, because transposons can increase in copy number during DNA replication
• A DNA transposon may be replicated, and then, still during the replication process, it may be cut and pasted into part of the genome that hasn’t been replicated yet, giving one ds daughter DNA molecule the same number of DNA transposons as before, but the second molecule one more copy of that DNA transposon
• When this happens, it is occurring during S phase of the cell cycle
• Draw out mechanism of DNA transposition, and the case of increasing copy of transposons
• DNA transposition is autonomous transposition
• However, in order for the process of transposition to work, the host cell’s machinery is still needed

Question 13

Retrotransposons and Retrotransposition

Answer 13

• Mobile elements of DNA that are able to move location in the genome via retrotransposition
• Retrotransposition follows a “Copy and Paste” mechanism by copying the mRNA of the retrotransposon, making DNA from it, and then inserting this newly made DNA into the target site

Retrotranscription:

• First, the retrotransposon is transcribed and translated into reverse transcriptase, integrase, and other proteins
• The reverse transcriptase then transcribes cDNA from the mRNA formed by the transcription of the retrotransposon, degrades the ssRNA, replicated cDNA into dsDNA, and then integrase inserts this newly made ssDNA into the target DNA

Question 14

Autonomous transposition

Answer 14

• mDNAs encode all the enzymes needed for transposition

Question 15

Telomeres

Answer 15

• DNA repeats at each end of both chromosomes that have no important genetic information
• In humans, telomeres are typically 10-15 kb long and are the sequence 5’ TTAGGG 3’
• Protects the ends of the chromosome from DNA damage by recruiting different proteins
• Helps prevent loss of important genetic information/material during replication

Question 16

Replication Problem: Loss of genetic information

Answer 16

• On the lagging strand, there is always an RNA primer that was put there to form the Okazaki fragment
• After the nuclease removes the RNA primers, we are left with a gap at the end, b/c DNA polymerase can’t come in and fill in the gap since it doesn’t have something to work off of
• This is why the cell makes these regions, the telomeres, have repeats of non-functional DNA sequences, b/c they can be lost overtime during DNA replication and not cause any loss of genetic information

Question 17

Telomerase

Answer 17

• Used to extend the length of the telomeres if they get too short
• It will bind to the end of the telomere and recruit a piece of RNA called “telomerase RNA”
• The telomerase RNA is complementary to the telomere, and it binds to the very end, so it has a repeated sequence hanging off that the telomerase makes the DNA from
• Since telomerase uses RNA as its template to make DNA, it is also a reverse transcriptase
• As the telomerase moves along, the telomerase RNA jumps forward with it to do the synthesis again and to keep extending the telomere region
• Draw out picture
• ## Telomerase is often up-regulated in cancer

Question 18

DNA polymeration vs RNA polymeration

Answer 18

• The monomer in DNA polymerization is dNTPs, and the monomer in RNA polymerization is NTPs
• Use DNA polymerase and RNA polymerase, respectively
• Helicase is used to separate the two strands; RNA polymerase is used to separate the two strands (DNA in both cases)
• Polymerization starts where the primer is located; Polymerization starts where the promoter is located, and doesn’t need a primer to start
• Both strands of DNA are the template; only one strand of the DNA is the template for each PROMOTER; there can be promoters on both strands, just coding for different genes
• Goes in 5’ —> 3’ in both
• Stops at the end of the template, or when it hits another DNA strand, like the lagging strand does; stops at the stop site, which is a DNA sequence
• The final product is 2 dsDNAs; final product is ssRNA
• Does 1 copy per cell cycle; can do multiple copies per unit of time (doesn’t have “cycles”)
• Replicates whole genome; only transcribes genes, which are specific regions of the genome that code for protein or RNA

Question 19

Promoter

Answer 19

• A sequence of DNA that the RNA polymerase recognizes as where to start transcription
• Tells the cell where to start transcription
• Tells the cell which strand to use as template
• Tells the cell how many copies of RNA to make, which is dependent upon how tightly the transcription factor binds.
• There can be different promoters on different strands, meaning each strand can act as the template strand and coding strand, they just differ in which one they act as depending on the gene
• As long as the promoter is the same sequence in the 5’—>3’ direction, the TF or RNA polymerase can bind, independent of whether its on the top or bottom strand
• Not all nucleotides in a promoter are important; there are small, functional elements in the promoter which are recognized and bound by transcription factors

Question 20

Enzymatic activities of RNA polymerase

Answer 20

• Can serve as polymerase
• Can serve as helicase
• Can serve as primase

Question 21

Transcription start site

Answer 21

• TSS for short
• The site where the first nucleotide is transcribed (added)
• Is labeled as +1
• DNA sequences after the transcription start site are labeled with + numbers deepening on how far away from the TSS they are
• DNA sequences before the transcription start site (including the promoter?) are referred to as “up stream,” and have a negative number assigned, depending on how many nucleotides away from the TSS they are
• THERE IS NO 0; we immediately go from -1 to +1
• Arrow that indicates the direction and where the TSS is is located on top of the coding strand, meaning that sequence that you see is what is going to be your RNA, but with U’s instead of T’s?

Question 22

Transcription factor

Answer 22

• A protein that recognizes the promoter sequence that RNA then binds to to initiate transcription
• I think in some cases, the transcription factor recognizes the promoter, not the RNA polymerase, and in some cases, the RNA polymerase directly recognizes the promoter

Question 23

E.coli’s promoter

Answer 23

• Has a transcription factor called the sigma factor, which binds to two regions upstream of the TSS, called the -10 box and the -35 box, since these are approximately where they are located in the genome in relation to the TSS

Question 24

Process of Transcription

Answer 24

• The transcription factor will bind to the promoter and recruit the RNA polymerase. Since the TF and RNA polymerase can only bind one way, this determines the template strand, and thus the direction of transcription
• The RNA polymerase will start synthesizing the RNA, and as it moves along it will dissociate from the TF, staying connected to the RNA at the 3’ end
• As the RNA is being made, it dissociates from the template strand it was made from because the extra -OH group at the 2’ carbon of the RNA prohibits it from making strong hydrogen bonds with the template strand
• Only the end attached to the RNA polymerase is still bound to the template strand

Question 25

How to determine which genomic sequences are genes

Answer 25

• Can’t just look at genomic genome
• Look at mRNA and match up mRNA sequences to DNA sequences
• Requires sequencing not only our genome, but also our mRNA

Question 26

RNA sequencing

Answer 26

• First add an RNA oligo to the 5’ cap of the RNA. The sequence doesn’t matter, it will just be used later on for primer design
• Then add reverse transcriptase and a primer with T’s (TTTTT…) to bind to the poly A tail of the RNA to make sure we start transcription in the right location
• Add RNase to degrade the mRNA so we are left with our cDNA
• Add a primer that is the DNA version of our oligo added earlier, since this will be complementary to the cDNA, and add DNA polymerase. We add the oligo to be able to have a DNA sequence we know that we can make a primer for to allow DNA polymerization of the entire gene to occur. If we didn’t have this oligo, we wouldn’t know the sequence of the 3’ end of our cDNA
• We now have a double stranded DNA copy of our mRNA, so we can insert this into a plasmid via ligation and then get the plasmid sequenced
• Essentially did reverse transcription, and then ligation

Question 27

Functional Elements in Promoters

Answer 27

• Not all nucleotides in a promoter are important for transcription factor binding
• Only specific, small (relative to the promoter itself) sections of nucleotides in the promoter are functional elements
• The functional elements are what are recognized and bound by the transcription factors
• There are two types of functional elements: common/general elements, such as the -10 box, -35 box, and TATA box, which are functional elements that exist in many different genes; the other type is gene specific functional elements, which exist only for their specific gene and no other genes
• Sometime it is useful to know how strong the promoter is, or in other words, how tightly it binds to its TF, and this can be determined via northern blot

Question 28

Northern Blot

Answer 28

• Can be used to detect how much of your RNA of interest is present, which can tell you how strong the promoter is, since the promoter strength determines how many copies of RNA will be made, and is thus directly correlated to how much RNA is present
• You will want to design a small DNA or RNA oligo that is complementary to a sequence in your RNA of interest, and label this oligo with a p32 radioactive label on its 5’ end. This will be your probe
• The first step is extracting and isolating out all of the RNA in the cell, and then running it on a gel to separate out the RNA based on size
• Then, transfer the RNA onto a nylon membrane from the gel. This is done so the RNA is exposed to the surface which is necessary for the next step. In the gel, it is actually inside the gel and not exposed to the surface
• Then , via a technique called hybridization, you want to attach your DNA probe to your RNA. This is done by placing the membrane in a solution containing your probe. The probe will bind to your RNA of interest and no other RNA, unless some other RNA has the same sequence complementary to your probe
• After removing the membrane, you can use the radioactivity to see which RNA is your RNA of interest, and, more importantly, how much of your RNA is present based on the intensity of the fluorescence of the probe, which indicates how strong the promoter is

Question 29

Transcription Reporter Assay

Answer 29

• A technique to determine strength of promoter
• Take promoter of interest and insert it in a vector, directly upstream of the plasmid’s “coding region of reporter gene.”
• Your promoter can then be used to make mRNA of the gene, which can then be transcribed into protein via the host’s machinery, and you can then measure the amount of protein produced
• To measure the amount of protein produced, this reporter gene is a gene that codes for something can itself be easily detected via eye, or a product of it can easily be detected
• Examples include luciferase, which emits a yellow light when interacting with luciferine, and GFP, which is protein that fluoresces green under UV light
• The amount of protein produced is indicative of how much mRNA was produced, which is indicative of how strong the promoter is

Question 30

Deletion series

Answer 30

• One of the techniques to determine the functional elements of a promoter
• Take the promoter and measure its activity (amount of RNA made/promoter strength) and set this as the baseline, at 100% activity
• Cut off a small piece of the promoter at the 3’ end and measure its activity, comparing to the activity of the intact promoter
• Keep subsequently cutting off pieces of the promoter and measuring activity, and when you get a significant decrease in activity, that tells you that the region you removed is part of or contains a functional element
• If you get an increase in activity after removing a section, that tells you the section removed is part of or contains the sequence where a repressor binds

Question 31

Linker Scan

Answer 31

• One of the techniques to determine the functional elements of a promoter
• First take the wildtype, intact promoter and measure its activity, setting this as 100%
• The introduce a mutation into a region of the promoter and measure its activity again
• Continue this, making mutations in different regions in the promoter
• Mutations that give activities significantly less than 100% were in regions that are important to promoter activity