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Biochemistry > Exam 3- L12 > Flashcards

Exam 3- L12 Flashcards

1
Q

What are two ways by which discrete segments of DNA can move from one region of the genome to another?

A
  1. Site Specific Recombination

2. Transposition (“jumping genes”)

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2
Q

What does site specific recombination require?

A

It requires a SPECiFIC short DNA sequence and an enzyme (recombinase).

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3
Q

Briefly explain Transposition.

A

Short sequences called transposons that can be excised and reinserted at a different place in the genome.

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4
Q

In Site Specific Recombination, where does the exchange between two DNA parental strands occur at?

A

It occurs at an extremely specific sequence, known as the recombination site.

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5
Q

What is present on both sides of a parental DNA strand during SSR?

A

Binding sites for the enzyme, recombinase. The directionality of the enzyme will inverted in direction.

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6
Q

What do the two DNA parental strands have to have?

A

They must contain the SAME EXACT CODE and they must have the same directionality.

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7
Q

How many total recombinases do you need to achieve this?

A

You need four recombinases. Two for each DNA parental strand.

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8
Q

What is a recombinase similar to and what does it?

A

It is similar to a topoisomerase.

It will bind the the DNA strand where it will cleave the phosphodiester backbone where recombination will take place and then it will be patched up.

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9
Q

What are the three outcomes of CSSR? (conserved site specific recombination)

A
  1. Integration
  2. Excision
  3. Inversion
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10
Q

Explain one way you would get integration from SSR.

A

You would have to have two circular DNA molecules going in the SAME direction, merging the two.

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11
Q

Explain one way you would get excision from SSR.

A

You would have one circular DNA with the recombination site in direct orientation (facing opposite directions), separating the two.

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12
Q

Explain one way you would get inversion from SSR?

A

You would have one circular DNA with the recombination site in inverted orientation (going in the same direction), inverted the sequence.

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13
Q

Does recombinase use ATP?

A

No, it is like Topoisomerase I.

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14
Q

What possible two AA can the recombinase have?

A
  1. Tyrosine

2. Serine

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15
Q

Explain the mechanism of the recombinase.

A

The tyrosine or the serine will perform a nucleophilic attack on the phosphodiester backbone, leaving you with different products.

Tyrosine: A free 5’ OH end

Serine: A free 3’ OH end.

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16
Q

Explain what happens after one attack of a recombinase.

A

After one nucleophilic attack of a recombinase, you are left with free 5’ or 3’ OH ends. These groups are nucleophilic in themselves, so they will attack the the opposite DNA strand, mixing the two.

This will repeat one more time to completely intertwine the sets of strands.

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17
Q

So what happens if two recombination sites are on the same DNA molecules and they are directionally facing each other? ->

A

You will have inversion.

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18
Q

So what happens if two recombination sites are on the same DNA molecules and they are directionally facing away from each other in opposite directions? -> ->

A

You will have deletion and insertion.

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19
Q

What is one application of SSR? (think gene expression)

A

There are specific sites within a gene where inversion or insertions can occur.

So image an operon that contains genes that are next each other, which are being expressed by a promoter sequence.

The promoter sequence is recognized by RNAP, where RNA production is occurring at those genes. The genes being produced are FLjB (flagella #1) and FljA (a repressor protein for FLjC).

When a Hin protein (recombinase) binds to its site, it will cause an inversion. This will disrupt the promoter and will turn OFF the production of FLjB and FljA.

Since FLjA was the one creating the repressor protein for FLjC, but it is turned OFF. You will have expression of the FLjC.

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20
Q

What are transposons?

A

Genes that can “jump” around, inserting themselves into a sequence. This can potentially disrupt a gene that is being expressed.

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21
Q

Explain the brief overview of the transposon mechanism.

A

You are going to have your transposon and your Target DNA in which you want to insert the transposon.

You cleave your target DNA with a transposase and then you insert your transposon at the site of the cut. (At the end of each transposon, you will have terminal repeats, which is your transposase binding site.

Once it is inserted, you fill in the gaps with replication (at the terminal repeats that were on the transposons) and then seal it up via ligase.

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22
Q

What are the two types of transposition reactions?

A
  1. Direct Transposition.

2. Replicative Transposition.

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23
Q

What are the four main steps of transposition?

A
  1. Cleavage
  2. Free ends of transposons attack target DNA.
  3. Varies
  4. Site-Specific Recombination.
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24
Q

Explain the four steps for direct transposition.

A
  1. Cleavage- you will have a double stranded break, going through both strands of DNA at the same area.
  2. The free 5’ OH/3’ OH end of the transposon will attack one strand of the target DNA and the other free 5’ OH/3’ OH end of the transposon will attack the other strand.
  3. Then you will have the gaps filled via DNA polymerase I and DNA Ligase.
  4. Site-Specific Recombination.
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25
Explain the four steps for replicative transposition.
1. Cleavage- you will have staggered cuts, one end of the transposon of one strand, and the the other end of the other strand. Producing 5'/3' OH overhangs. 2. Then you will have nucleophilic attack with both of the 5'/3' OH ends will attack the circular target DNA, creating this hybrid-type of intermediate. 3. Then the whole transposon will be replicated via DNAP III, creating cointegrate strands. 4. Once you have pairing, you will end up with one straight DNA strand and one circular DNA strand, each with a segment of both the transposon and the newly synthesized DNA.
26
What is a use of SSR in real life?
The production of antibodies.
27
How is SSR used in adaptive immunity?
Antibody genes are able to maximize protein diversity with the limited amount of genetic information that they have. DNA rearrangement (SSR) during assembly of the antibody genes accounts for this diversity.
28
What are two main structures of an antibody?
Light chain- composed of 3 genes Heavy chain- composed of 4 genes.
29
Explain how the variable of antibodies comes to be.
In an immature B-cell, you are going to have a variety of genes present. In the process of being a mature B-cell, the B-cell will have to choose which specific segments it needs for the production of antibodies. In light chains: ``` V = Variable Segment J = Joining Segment C= Constant Segment ``` Heavy chain (has an extra variable, D = diversity segment). Each of these variables have a subclass (i.e. V1, V2, v3), but only one of them can only be chosen for the end result of the mature B-cell. Once you recombine each of the variables into a VJC region, it can be scribed and translated for that specific gene. The removal of the extra variables is via SSR, creating deletion.
30
Explain the steps for the production of the segment for the antibody.
1. Cleavage of the intervening DNA via RAG1 and RAG 2. These enzymes recognize RSS sites (recombination signal sequences). Cleavage produces 5'/3' OH over hangs. 2. Nucleophilic attack on its own sister strand, producing a deletion via transesterification. 3. Double Stranded Break Repair via non homologous end joining will merge together the two variables.
31
What is the molecular dogma?
DNA -> RNA -> Protein DNA -> RNA : Transcription RNA -> Protein: Translation.
32
What is unique about RNAP in prok?
Only a single RNAP makes all of the RNA's. This is different from eukaryotes.
33
What does a RNAP require?
1. DNA Template 2. Mg2+ 3. rNTP
34
What is holoenzyme?
Core Enzyme + Sigma Subunit
35
What is the sigma subunits role?
It controls where the RNAP goes. Specifically, to the promoter sequence.
36
What is the 5-Step Transcription cycle?
1. Closed Complex 2. Open Complex 3. Initiation 4. Elongation 5. Termination
37
Rho-dependent terminates how?
It forms a hairpin structure followed by a stretch of Uridine (U).
38
What is a gene?
A segment of DNA that contains all of the information needed for regulated synthesis of RNA or protein product.
39
What features of an mRNA are present within its gene.
Promoter Sequence Shine-Delgarno- RBS in prokaryotes. Ribosome Binding Site Coding Sequence Terminator Sequence
40
What is the shine-delgarno?
It is the ribosome binding site in prokaryotes. It is an A-G rich sequence. AGGAGG is the sequence.
41
What is unique about the 5' end of an RNA strand?
It retains all three of its phosphate groups; all subsequent nucleotides release PPi when added to the chain and only retain the alpha phosphate.
42
Transcription always occur in what direction?
5' -> 3' Direction
43
What is the significance of the sigma factor?
It is important in finding the promoter sequence so that RNAP can bind to DNA.
44
Describe RNAP in terms of landmarks and structures.
RNAP has many different entrances and exits. You have an exit site for your newly synthesized RNA. You have an entrance for your DNA molecule. You have an entrance for you rNTP. It has an active site with Mg 2+ present.
45
What is the rudder?
It is a piece of protein that sticks into dsDNA and unzips the DNA molecule as RNAP is moving. This is the helicase of RNA and it does not require ATP.
46
What does RNAP read?
It only reads one strand, and it is going to be the 3' -> 5' end.
47
Describe the active site of RNAP.
1. DNA template (only one strand will be copied) 2. Substrates of rNTP 3. Divalent Ions (Mg 2+) You will also have Asp, stabilizing the charges of the Mg 2+.
48
Describe the DNA molecule as it is entering and then leaving RNAP.
Before the DNA strand is entering RNAP, it is positively supercoiled. Then it will reach the rudder where it will unwind (~17BP). After that, DNA will twist into a negative supercoil.
49
What structure does the rudder make?
Transcription bubble.
50
What are three names that can be used to describe the strand being used in direct contact when synthesizing RNA? What about the other?
Template, Non-coding, Anti-Sense Strand Non-template, Coding, Sense Strand
51
What is +1? What about the other numbers?
+1 is the Transcription Initiation Site (TIS). It is the place where the first NT will be deposited during transcription. Anything upstream of that, meaning comes before that site, is a negative number. Anything downstream of that, meaning comes after the site (direction it is moving in), is a positive number. There is no ZERO!
52
What is a promoter?
It is a sequence that guides RNAP to the transcription initiation site.
53
How would you map a promoter?
1. First you would produce identical DNA fragments and radioactively label one end of one strand. Then you would separate them into two separate tubes. One with RNAP present and the other without (control). 2. Then you would add in DNase I, which is a restrictive exonuclease. It will only cut the DNA strand once and it will not cut at an area where RNAP is bound. It makes random cuts. 3. Then you would isolate the DNA fragments and denature any proteins attached to it (i.e. RNAP). Only the labeled fragments will be present in the next step. 4. You then run the fragments on a polyacrylaminde gel electrophoresis and visualize the radiolabeled bands. So since the cuts were random and was never cut at an area where RNAP was bound, missing bands indicate where RNAP was bound to DNA.
54
Where does RNAP bind to?
RNAP binds to a region of DNA from -40 to +20.
55
Describe what upstream from the initiation site looks like.
TTGACA (-35) -> {~18NT) (Spacer) -> TATAAT ("-10) -> {~8NT) -> Initiation Site
56
What is a consensus sequence?
It reflects the predominant nucleotides at which position on DNA, RNAP will bind to. Website definition: The consensus sequence is the calculated order of most frequent residues, either nucleotide or amino acid, found at each position in a sequence alignment.
57
What are some promoter features?
The closer the match to the consensus sequence, the stronger the promoter (-35 and -10 boxes). The absolute sequence of the spacer region (between the -35 and -10 boxes) is NOT important, but the LENGTH of the spacer sequence IS important.
58
Do you think that all genes in E. Coli have a promoter sequence that is recognized by sigma 70?
No, not all genes in E. Coli have a Sigma-70 promoter sequence. Different sigma factors bind the core polymerase to form holoenzymes with different promoter-binding specificities. Example: Heat Shock Response -> high temperatures induce the production of Sigma-32, which binds to the core polymerase to form a unique holoenzyme for recognition of promoters of heat-shock genes.
59
How do you know with promoter is used?
The relative concentrations of sigma factors and their relative affinities to RNAP will dictate which promoter will fire.
60
What constitutes a holoenzyme?
Sigma Factor + Core Polymerase
61
Compare and contrast the activity of just the core enzyme (3) vs. the holoenzyme (4).
Core Enzyme: - No specific promoter binding (sigma is absent) - Tight non-specific DNA binding - Weak polymerase activity from various start sites Holoenzyme: - Binds to specific promoters - Weak non-specific DNA binding - Finds the promoter 10,000x faster - High polymerase activity.
62
What is a sigma factor? What is unique about them.
It is a protein need for initiation of RNA synthesis. Within sigma factors, you will have several domains (i.e. Sigma 1.2, 2.6, etc). Each domain has a different function and they are all connected to each other by flexible linkage.
63
What is the role of the Sigma-1 domain?
Sigma-1 mimics a DNA strand. It has a lot of NEGATIVELY CHARGED AA associated with it. So as the holoenzyme is scanning the DNA, the Sigma-1 domain sites inside RNAP, acting as dsDNA. When RNAP finds its promoter sequence and forms an open complex, the Sigma-1 domain will dissociate to make room for DNA.
64
What is the role of the Sigma-2 domain?
Sigma-2 interacts with the -10 Region and unwinds DNA. It is the region responsible for changing the DNA strand going from an closed complex to an open complex (transcription bubble formation). Unlike in DNAP, it DOES NOT need a helicase or ATP to achieve this. The Sigma-2 region has AA that interact with the nucleotides within the -10 region. Since they are similar in structure, the NT (A & T) on the non-template strand will flip to negate base-pairing between the two. This promotes melting of DNA (melting = unwinding). Note: This is before initiation has occurred. You are forming this open complex first so that RNAP can position itself correctly. The rudder will be the one unzipping it post-initiation.
65
What is the role of the Sigma-3 Domain?
Allows for initiation to occur. Initiation of transcription requires a RNA molecule to to produced. Eventually, the newly synthesized RNA has to exit ENAP through the RNA exit channel. When the RNAP starts transcription, it will start to go, then it will stop, go, stop. The Sigma-3 loop is blocking the exit channel of the RNA strand. Once this structure is removed, this will truly be the start of transcription. It needs a lot of energy to do this.
66
What is the role of the Sigma-4 Domain?
It recognizes the -35 region. It is going to strongly make contact and bind to the -35 region. It has an alpha helical structure and one of the helices will sit in the major groove at the -35 region, stabilizing the complex.
67
What are the 6 basic steps of the RNAP transcription cycle in prok?
1. Holoenzyme binds to the promoter. (closed complex) 2. Transcription bubble formation (open complex) 3. Absorptive Initiation 4. Sigma-70 dissociates after transcription has officially begun. 5. Elongation 6. Termination
68
What structure is involved in abortive initiation?
Prokaryotes: Sigma-3 Factor Eukaryotes: TFIIB Both of these proteins have a loop that extends within the RNAP active site, inhibiting transcription. The loop must be removed in order for elongation to occur.
69
What does the displacement of the loop called?
Promoter clearance. It is thought to help the polyermase break away from the promoter sequence to continue onward.
70
What happens during promoter clearance?
RNAP undergoes a conformational change that associates with DNA very stably and loosens its grip on the initiation factors/sigma factors
71
What would you expect to happen to RNAP if the -10 region of the promoter is altered in such a way that it can't be recognized by the RNAP holoenzyme?
RNAP can find the bind to the promoter sequence (because the -35 region is still normal), but it can't initiate transcription.
72
What happens at the termination sequence?
RNAP will read THROUGH the sequence.
73
What are two ways in which transcription is terminated in prokaryotes?
1. Rho-Dependent Transcription Termination | 2. Rho-Independent Transcription Termination
74
Describe Rho-Dependent Transcription Termination.
It needs a protein (rho) AND a DNA sequence (RUT. When RNAP uses rho-dependent termination (with rho being the protein), rho binds to an area of the RNA that is rich in cytosine, called the RUT sequence. The rho-protein, acts like a helicase and through ATP hydrolysis, it pulls out the RNA strand that is associated with RNAP.
75
RUT sites are rich in what?
Cytosine- C-Rich
76
What direction does the rho move in?
It moves in the same direction as synthesis, 5' -> 3' on the RNA strand.
77
Describe Rho-Independent Transcription Termination.
Within the RNA that is being produced, a hairpin structure is going to be made, termed the terminator hairpin. Next to the hairpin in the RNA, you will see a whole bunch of U's that is going to be transcribed due to the A-T rich zone on the DNA strand. The U-A interaction between the RNA strand and the DNA strand is weak (because there are only 2-Hydrogen bonds), the RNA will dissociate from DNA.
78
What do you need in order to form a hairpin?
You need dyad symmetry. Dyad symmetry refers to two areas of a DNA strand whose base pair sequences are inverted repeats of each other. They are often described as palindromes. You need these sequences because they will bind to each other to form the hairpin.
79
What happens after the hairpin has formed?
The polymerase will pause. If the hairpin is a true terminator, RNA will dissociate from the DNA strand as the A-U base pairing is unstable.
80
What is Rho-independent dependent on?
Depends on dyad symmetry (for the formation of a hairpin) followed by U's.

Biochemistry (17 decks)

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