Recombination

Question 1

What is the RecBCD pathway?

Answer 1

It is a repair system for double stranded breaks in the DNA. It uses homologous recombination for repair.

Question 2

How can a single stranded nick lead to a collapsed replication fork?

Answer 2

As the fork moves across the chromosome, a nick in the parental double strand will disrupt the replication and lead to a double stranded break.

Question 3

What is the function of the RecB protein?

Answer 3

3’-5’ helicase and nuclease

Question 4

What is the function of the RecC protein?

Answer 4

Recognizes chi sites

Question 5

What is the function of the RecD protein?

Answer 5

5’-3 helicase

Question 6

How does the RecBCD system work?

Answer 6

RecBCD complex binds onto double strand of the collapsed replication fork, and RecA scans for homologous sites in the

Question 7

In lambda red recombination, why is Gam protein crucial?

Answer 7

Gam protein will inhibit the RecBCD complex from binding to the PCR product and start the degrading until it finds a chi site. There are no chi sites in the PCR product and RecBCD will therefore continue the degrading until there are only fragments left.

Question 8

What is the purpose of the Exo protein in lambda red?

Answer 8

Exo has nuclease activity, binds to the dsDNA PCR product and degrades DNA in 5’-3’ direction, leaving ssDNA overhangs.

Question 9

What is the purpose of the Bet protein in lambda red?

Answer 9

The Bet protein binds to the ssDNA overhangs that were produced by Exo, and protects the linear DNA from being degraded by the cellular degrading systems. Bet also promotes annealing of the single strands to the complementary single strand in the target DNA sequence. Bet faciliates exchange and recombination between the target gene and the PCR fragment.

Question 10

How does transposons speed up evolution?

Answer 10

Transposons jump randomly in and out of genes and therefore cause random alterations.
They can jump into a gene and inactivate it.
If two identical transposons are flanking a set of genes, they can cause duplications and deletions in homologous recombination.

Question 11

What is MMR (mismatch repair)?

Answer 11

MMR is a repair system for mismatches between base pairs

Question 12

What is tautomerization and how can it cause replication errors?

Answer 12

A position change of a proton within a base, yielding a change in chemical formula which alters the base pairing rules. This lets bases pair with the wrong bases –> wrong base is incorporated into daughter strand –> point mutation.

Question 13

Give an example of a tautomerization in DNA.

Answer 13

A thymine in its keto form base pairs with an Adenine, as normal. But if tautomerization occurs the thymine could switch to its enol form –> it can now pair with G.

Question 14

Which are the purines? Single or double ring?

Answer 14

Adenine and guanine. Double ring.

Question 15

Which are the pyrimidines? Single or double ring?

Answer 15

Thymine, cytosine and uracil. Single ring.

Question 16

What is a transition?

Answer 16

A change of base I the same class, for example purine –> purine.

Question 17

What is a transversion?

Answer 17

A change of base in between classes , for example purine –> pyrimidine.

Question 18

Why are transitions more common than transversions even though there are more combinations of transversions?

Answer 18

Transition-caused mutations are generated at a higher frequency because it is more likely that it’s a change from single-ring to single ring or double ring to double ring.

Question 19

What are the characteristics of a gram positive cell wall and how is it stained?

Answer 19

Thick peptidoglycan layer which makes up the thick cell wall. Only inner membrane. Low lipid content.

When staining with crystal violet, most of the dye stays when washed away with ethyl alcohol and the cells receive a violet color.

Question 20

What are the characteristics of a gram negative cell wall and how is it stained?

Answer 20

Thinner peptidoglycan layer that makes up the cell wall. Inner and outer membrane flanking the cell wall. Higher lipid content than gram positives, but no teichoic acids.

When stained with crystal violet and washed with ethyl alcohol, most of the dye is removed. But recieve a pink color when dyed with safranin afterwards.

Question 21

Are gram positives or gram negatives more susceptible for antibiotics?

Answer 21

Gram positives.

Gram negatives have this outer membrane which contains both lipids and porins and other proteins. Antibiotics have to pass through the membrane to reach their target.

Since the outer cell membrane of gram negatives contains a wide range of different properties, it also has the possibility to produce alterations in these properties to make them untargetable for antibiotics –> confer resistance.

Question 22

What properties of the bacteria does beta-lactams target?

Answer 22

Beta lactams are bactericidal and targets the cell-wall formation by inhibiting the cross-linking of the peptidoglycan layers. They do this by binding covalently to enzymes that are essential for the formation of the cross-links.

Question 23

What is the genome size of the E. coli chromosome (in base pairs)?

Answer 23

4,6 *10^6 bp

Question 24

What are the three main regulatory systems for replication initiation?

Answer 24

OriC sequestration with SeqA
datA binding of DnaA
RIDA (Regulatory Inactivation of DnaA)

Question 25

How does OriC sequestration with help of GATC sites help inhibit early initiation of replication?

Answer 25

GATC-sites are targeted for methylation. Although, newly synthesized strands are not methylated yet. The protein SeqA recognizes non-methylated strands and binds to them --> also binds to inner membrane of cell to "hide" the DNA from DnaA binding to early --> inhibiting the DNA from being replicated again for a while after replication.

Question 26

What is negative regulation of a gene?

Answer 26

A system where a repressor binds to the gene in order to repress gene expression. In this case, the gene is usually normally turned on (when repressor is not binding)

Question 27

What is positive regulation of a gene?

Answer 27

A system where an activator binds to the gene in order to activate gene expression. In this case, the gene is normally repressed (when the activator is not binding)

Question 28

Explain how a negative inducible system works. Catabolic or biosynthetic operons?

Answer 28

A repressor normally binds to the promoter - inhibiting gene expression. But in presence of an inducer, the inducer may bind to the repressor --> repressor will release from the promoter and gene will get expressed. Usually catabolic operons (substrate is converted to carbon and utilized as energy source). The substrate is the inducer for the operon, i.e only activated in presence of the substrate.

Question 29

Explain how a negative repressible system works. Catabolic or biosynthetic operons?

Answer 29

A repressor normally does not bind to the promoter - gene expression is activated. But in presence of a corepressor, corepressor binds to repressor --> binds to promoter --> represses gene expression. Usually biosynthetic operons. The enzymes encoded by the gene are involved in biosynthesis of certain compounds. The corepressor is usually the end compound or a biproduct - this represses the system when the end product is already present.

Question 30

Explain how a positive inducible system works. Biosynthetic or catabolic operon?

Answer 30

An activator is normally not bound to a promoter, keeping the gene turned off. In presence of inducer --> gene is expressed. Usually catabolic operon. The substrate is the inducer for the operon, and broken down and utilized as a carbon source. The gene expresses enzymes ivolved in the breakdown of the substrate.

Question 31

Explain how a positive repressible system works. Catabolic or biosynthetic operon?

Answer 31

An activator is normally bound to the promoter, keeping the gene activated. A corepressor can bind to the activator and repress gene expression. Usually biosynthetic operons. Enzymes encoded by the gene are involved in synthesizing certain compounds, and the corepressor is usually the end compound or a biproduct.

Question 32

What are the subunits of RNAP?

Answer 32

B B' alpha alpha' omega These all constitute the core enzyme. With the additional sigma factor which is only needed for initiation, it's called the holoenzyme.

Question 33

Describe the process of intrinsic transcription termination (factor-independent)

Answer 33

Intrinsic termination occurs when the RNAP transcribes a transcript containing two inverted repeats close to each other --> they form a hairpin loop by base pairing with each other. the hairpin is directly followed by a poly-U, which aids the RNAP in releasing from the transcript

Question 34

Describe the process of rho-dependent termination.

Answer 34

RNAP transcribes a rut (rho utilization) site which the hexameric protein rho binds to and starts to chase RNAP. Soon enough RNAP encounters a rho pause site and stalls, which allows rho to catch up and release RNAP from the transcript.

Question 35

Describe how traditional cloning is done?

Answer 35

Done with restriction enzymes. It's preferable to use restriction enzymes that cut at the sites exactly where you want your insert in the plasmid. The insert should be flanked with the sites that match the backbone. The gene is inserted into the cell with the plasmid and ligase glues the pieces together.

Question 36

Describe how gateway cloning is done?

Answer 36

Gateway cloning is facilitated by lambda red enzymes. Homologous sites at the plasmid and the insert genes are created, and lambda red enzymes are expressed to perform homologous recombination.

Question 37

Describe how TOPO cloning is done?

Answer 37

TOPO cloning is topoisomerase dependent cloning Insertion gene is amplified and Taq polymerase is used to add A (deoxyadenosine) overhangs to the insert. Complementary T overhangs in the plasmids are synthesized by Topoisomerase. The gene is then inserted into the plasmid and ends are rejoined by Topo.

Question 38

What is pegRNA?

Answer 38

Prime editing guide RNA. Involved in prime editing and consists of a complementary strand to the target DNA (lower part of the RNA) and a strand containing new genetic information that is to be incorporated into the DNA (upper part of RNA).

Question 39

What is the function of Reverse transcriptase in prime editing?

Answer 39

Reverse transcriptase uses RNA as a template to transcribe DNA. The upper part of pegRNA has a sequence complementary to the DNA strand that is to be edited, and that part is followed by new genetic information. pegRNA binds complementary to DNA and the new genetic information is reversibly transcribed into the ssDNA.

Question 40

What is a major drawback of prime editing?

Answer 40

The repair systems in the cell treat the incorporation as mismatches, and the edit will be deleted at a certain frequency.

Question 41

What is Cas13 used for?

Answer 41

Cas13 binds and cleaves RNA instead of DNA. Is used to make non-permanent changes and is beneficial when avoiding the consequences of off-target mutations, because these mutations are not very problematic in RNA.

Question 42

What are the main types of spontaneous DNA damage (non-induced)? And what causes the mutation for each type?

Answer 42

Deamination of bases - changes base pairing rules Alkylating and oxidating agents - causes DNA base damage - could cause several different mutations UV damage - causes pyrimidine dimers - interpreted as a single base, leads to -1 frameshift mutation

Question 43

How does UV light induce mutations?

Answer 43

The conjugated ring in pyrimidines is extra absorbent of light in UV wavelenghts. The absorbed photons energize the electrons and causes the double bonds to bind differently --> formation of a dimer between two pyrimidines. This dimer could be interpreted as a single base by the DNA Pol, which causes a -1 frameshift mutation.

Question 44

How does deamination cause mutations?

Answer 44

Deamination of bases is caused by deaminating agents and results in loss of the amino group in the base --> alters the base pairing rules.

Question 45

What are some repair mechanisms for deamination of bases?

Answer 45

Methyl directed Mismatch repair: Mut complex recognizes mismatch in DNA and replaces it with the correct base. Is dependent on the methylation of strands in order to recognize the newly formed strand DNA glycosylases: enzymes that break the bond in between the sugar backbone and the damaged base

Question 46

Describe the mechanism of MMR?

Answer 46

MutS recognizes and binds a mismatch. MutS recruits MutL. About a kilo bp away, MutH binds to a hemi-methylated GATC site. MutS, MutL and MutH form a complex,, creating a loop in the DNA. MutL creates a nick in the strand with the mismatch at the GATC site - and UvdR helices unwinds the helix so that exonucleases can start to degrade the strand until the mismatch is reached. DNA Pol III starts to re-synthesize the strand from the GATC site until the right nucleotide is incorporated at the mismatch DNA ligase ligases the strand together.

Question 47

Describe the structure of DNA Pol III holoenzyme.

Answer 47

The holoenzyme consists of 3 main parts: the core enzyme, the Tau subunit and the clamp loader. Corę enzyme: two alpha, two epsilon and two theta. Alpha subunits: has the main catalytic responsibility in polymerisation of new strand. Increases epsilon's affinity for the 3'-OH end at the DNA strand. Epsilon subunits: 3'-5' exonuclease activity, proofreads the newly synthesised strand and repairs mismatches. Clamp loader: dissociates the two mother strands during replication

Question 48

What are the benefits of lysogenic phage growth?

Answer 48

If the lytic phase id very effective, the process of using and lysing hosts might be too fast which could lead to all hosts dying, leaving the phages without a way to replicate.

Question 49

What is an example on how we can use phages in the lab?

Answer 49

We can use them for transducting bacterial genome from one cell to another.

Question 50

What is wobble?

Answer 50

The third position of the codon can does not have as precise pairing as the other two, permitting a single tRNA to recognize more than one codon. For example, Inosine in the codon can pair with both A, U and C in the anticodon.

Question 51

What is the purpose of wobble?

Answer 51

Because each tRNA can recognize more than one type of codon, the pool of tRNA does not have to be vary large, i.e there doesn't have to be one specific tRNA for each codon. Flexibility in the last position often encodes for the same amino acid, meaning that mutations in this position often doesn't have dire consequences. Wobble also optimizes elongation because it makes it more effective, i. e there are more tRNAs available for each codon.

Question 52

How does codon usage bias optimize elongation?

Answer 52

codon usage bias is that specific codons are used in a higher frequency than others, e.g two codons that code the same amino acid are not used at the same frequency. This makes translation faster and more effective because all tRNAs does not have to be produced in the same amount. During elongation, the incoming tRNA is randomly sampled on the codon since the cell doesn't "know" which is the right anticodon. Therefore, a larger pool of the tRNAs that are more used increases the likelihood that it will match the codon --> makes the translation faster.

Question 53

Describe the initiation of translation

Answer 53

Ribosome arrives to the mRNA and binds the SD sequence, the 16SrRNA is aligned with the SD --> the ribosome is located to the start codon. The first tRNA that arrives carries a formulated methionine, which sits on the P site rather than the A site.

Question 54

What is the purpose of the shine dalgarno sequence and where is it located?

Answer 54

It is located upstream of the Transcription initiation region. The purpose of the sequence is to help the ribosome align itself on the correct initiation codon.

Question 55

What factors are active in the initiation of translation.

Answer 55

Initiation factors 1-3 (IF1-3)

Question 56

What is the purpose of IF1 in translation initiation?

Answer 56

It binds to the A site to prevent the initiation tRNA from binding there.

Question 57

What is the purpose of IF2 in translation initiation?

Answer 57

It facilitates the transportation of the initiator tRNA onto the P site, and promotes the initiator complex (mRNA, 30S subunit and fMet-tRNA)

Question 58

What is the purpose of IF3 in translation initiation?

Answer 58

Binds to the E site and prevents premature binding of the 50S.

Question 59

Describe the steps of elongation in translation

Answer 59

A new tRNA with an aa is located onto the A site, and the peptide chain on the tRNA in the P site is transferred onto the new tRNA with the amino acid in a peptidyltransferase reaction. EF-Tg pushes the ribosome along the mRNA. The tRNA in the P site moves to the E site and exits. Ef Tu catalyses the binding of a new tRNA-aa to the A site.

Question 60

How does termination work in translation?

Answer 60

A release factor binds to the A site. which causes the complex to disassemble.

Question 61

What is ribosome rescue?

Answer 61

There are cases where the stop codon is absent from the mRNA getting translated. This could be because the RNA has gotten cleaved, the transcription was terminated early or a mutation has caused the removal of the stop codon. In this case, the translated protein will be defective and maybe toxic. tmRNA is a combination between tRNA and mRNA, and has a coding sequence coding for additional proteins and a stop codon. The coding sequence is transcribed

Question 62

What could be a problem during protein folding? What is a solution?

Answer 62

Exposed hydrophobic amino acids in new peptide chains can associate with other hydrophobic aa:s in other proteins before the protein is folded --> aggregation. Chaperones bind to these areas and cover them while they're exposed. Chaperonins create a stable environment for the protein while folding.

Question 63

Describe the regulation of the ribB gene. Negative or positive regulation?

Answer 63

ribB gene is under negative regulation. ribB encodes an important intermediate in the riboflavin synthesis, and some of the riboflavin is then broken down to the compound FMN, which acts as a repressor in the system. In absence of riboflavin in the cell, the promoter of ribB is not repressed and the gene is expressed. In presence of riboflavin, some of the compound is broken down to FMN --> prevents transcription.

Question 64

How does the ribB riboswitch work?

Answer 64

The ribB gene riboswitch is located in the 5'-UTR of the transcribed mRNA of the ribB gene, and the byproduct FMN of riboflavin works as a ligand in the system. The riboswitch works on both a transcriptional and translational level. FMN binds to the aptamter upstreams of the region where the termination loop forms. When ligand binds, the terminator structure is formed. The structure is followed by a poly-U, which aids the RNAP in releasing from the transcript. In absence of ligand, the terminator structure does not form. But in cases where the RNAP manages to overcome the terminator structure, the terminator structure can sequester the RBS, which prevents the protein from getting translated even though the gene is transcribed.

Question 65

What are the main types of pre-translational regulation (before initiation)?

Answer 65

Riboswitches - sequestering of the RBS Regulation by sRNAs (small non-coding RNAs) Thermosensors - heat induces formation of anti-termination structure --> translation

Question 66

What are the main types of post translational regulations?

Answer 66

Posttranslational modification - for example two component systems (phosphorylation) Turnover - regulation by proteolysis: during normal conditions, Rssb protein is expressed which breaks down the protein. But the protein is not broken down during stress conditions

Question 67

What is the major difference between the major and minor groove in the helix?

Answer 67

Major groove is wider than minor and carries more detectable "code points" (hydrogen atom, H-bond acceptor and donor, methyl groups) of the base pairs. Therefore, each base pair combination has it's unique combination of code points, making it possible for protein to tell exactly what type of bond it is.

Question 68

Describe the process of replication.

Answer 68

DnaA binds to R1-R5 binding sites, inducing negative supercoiling at these sites. This induces helix opening at the DUE sequence upstream (AT rich = weaker bonds) when DnaC delivers DnaB to DUE and DnaB induces unwinding. Formation of replication bubble and SSB proteins bind to the single strands to keep them from winding again. DnaG primes the strand with the first RNTs (RNA sequence) because RNA Pol III cannot start without a primer. Then RNA Pol III recruited and initiates elongation. One leading and one lagging strand (lagging strand is made up of Okazaki fragments that are ligated together). Helicase unwinds the helix as the fork moves forward.

Question 69

How does tetracycline target bacteria?

Answer 69

Tetracyclines irreversibly bind to the A site in the ribosome-mRNA complex, inhibiting initiation of elongation. Most often are they bacteriecidal, because they inhibit replication and proliferation.

Question 70

What types of resistance mechanisms have been found in bacteria against tetracyclines?

Answer 70

Drug efflux Structural change in ribosome so tetracycline cannot bind Enzymatic inhibition of tetracycline molecule

Question 71

Describe the process of transcription.

Answer 71

Together with sigma factor, the RNAP is the holoenzyme. Sigma factor recognizes specific set of promoters, subunit 4 binds to -35 region and subunit 2 binds to TATAAT box. Beta' pincher wraps around the DNA strand to form the active site. the sigma factor separates the strands at the TATAAT box and and binds the coding strand. Elongation stage starts when the sigma factor has left the RNAP and RNAP leaves the promoter.