Microbial genomics exam 1

What is the difference between a nucleoside and a nucleotide?

A nucleoside is made of a sugar and a nitrogenous base only. It does not contain any phosphate groups.
A nucleotide is a nucleoside that has one, two, or three phosphate groups attached.
This difference is important because nucleotides are the building blocks of DNA and RNA, while nucleosides are incomplete units.

What sugar is in an RNA nucleoside?

RNA nucleosides contain ribose sugar.
Ribose has a hydroxyl (–OH) group at the 2′ carbon, which affects RNA’s structure and stability.

What sugar is in a DNA nucleoside?

DNA nucleosides contain deoxyribose sugar.
Deoxyribose lacks the 2′ hydroxyl group and instead has hydrogen, which makes DNA more stable.

What shape does the ribose sugar have when we draw it?

Ribose is drawn as a five-membered ring (pentose ring).
This ring shape allows bases and phosphates to attach in specific positions.

What is the major difference between an RNA and a DNA nucleoside?

The major difference is at the 2′ carbon:

• RNA has a 2′- OH group

DNA has H instead of OH
This difference makes RNA more reactive and DNA more stable.

Why are the carbons on the ribose sugar labeled 1′, 2′, 3′ instead of 1, 2, 3?

They are labeled with primes (′) to distinguish sugar carbons from base carbons.
Bases are numbered without primes, so this prevents confusion when studying structure.

What does NMP, NDP, and NTP stand for?

• NMP = Nucleotide Monophosphate (1 phosphate)

• NDP = Nucleotide Diphosphate (2 phosphates)

• NTP = Nucleotide Triphosphate (3 phosphates)

What does dNMP, dNDP, and dNTP stand for?

• dNMP = Deoxynucleotide Monophosphate

• dNDP = Deoxynucleotide Diphosphate

• dNTP = Deoxynucleotide Triphosphate

What bases are in DNA?

DNA contains:
Adenine, Guanine, Cytosine, Thymine.
These bases form complementary pairs.

What bases are in RNA?

RNA contains:
Adenine, Guanine, Cytosine, Uracil.
Uracil replaces thymine in RNA.

What is the difference between uracil and thymine?

Thymine has a methyl group, uracil does not.
This makes thymine more stable and better for long-term storage.

Which phosphates are alpha, beta, and gamma?

• Alpha: closest to sugar

• Beta: middle

• Gamma: farthest

Which sugar positions are connected in nucleotides?

The 3′ carbon of one sugar connects to the 5′ carbon of the next.
This forms phosphodiester bonds and creates directionality.

Which bases are purines?

Adenine and Guanine.
They are larger and have two rings.

Which bases are pyrimidines?

Cytosine, Thymine, Uracil.
They are smaller and have one ring.

 Which bases form base pairs?

A pairs with T (or U).
G pairs with C.

Which bases have two rings?

Purines

Which bases have one ring?

Pyrimidines

How many hydrogen bonds in A–T?

2

How many hydrogen bonds in G–C?

3

What is the major hydrogen bond interaction?

Between an amine group and an oxo group.
This stabilizes base pairing.

What reaction does ribonucleotide reductase do?

It removes the 2′-OH group to make deoxyribonucleotides.
This supplies DNA building blocks.

Which nucleotide is not reduced?

dTDP.
Thymine is made by a different pathway.

What does a kinase do?

Adds phosphate groups using ATP.
This activates nucleotides.

If kinase acts on GDP, what is formed?

GTP.
One phosphate is added.

What does a phosphorylase do?

Removes phosphate groups.
This reverses kinase activity.

What does thymidylate synthase do?

Converts dUMP into dTMP.
This produces thymine.

Why is thymidylate synthase important?

It is the only way to make thymine for DNA.
Without it, DNA cannot be synthesized.

What does THF do?

Carries methyl groups.
These are needed for thymine synthesis.

In what direction are sequences written?

5′ to 3′.
This matches synthesis direction.

Why are DNA strands antiparallel?

One strand runs 5′→3′, the other 3′→5′.
This allows proper base pairing.

Why is DNA more stable than RNA?

DNA lacks 2′-OH and is double stranded.
These features reduce breakdown.

Difference between major and minor grooves?

They differ in size.
Proteins bind mainly to the major groove.

What does DNA polymerase need?

Template, primer, 3′-OH, Mg²⁺.
All are required for synthesis.

What groups are involved in nucleophilic attack?

3′-OH attacks alpha phosphate.
This forms phosphodiester bonds.

Why is pyrophosphatase important?

It removes PPi, driving the reaction forward.
This makes replication irreversible.

Direction template is read?

3′ to 5′.
So synthesis goes 5′ to 3′.

Why is 3′→5′ exonuclease important?

It removes mistakes.
This increases accuracy.

 DNA Pol I and III activities

• Both: synthesis and proofreading

• Only Pol I: primer removal

What does helicase do and where does energy come from?

It unwinds DNA using ATP.
ATP provides energy.

What is SSB and what does it do?

Single-Strand Binding protein.

Keeps strands apart.

Why is DnaA important?

It starts replication.
It binds oriC and opens DNA.

What does primase do?

Makes RNA primers.
Polymerase needs primers to start.

Why is ligase important?

It seals DNA breaks.
Without it, fragments stay separate.

What groups are needed to connect DNA?

5′ phosphate and 3′ hydroxyl.
Ligase joins them.

What enzymes add phosphate?

Kinases

Replication fork

Contains helicase, primase, SSB, Pol III, Pol I, ligase, clamp.

What makes up the primosome?

DnaB and DnaG.
They unwind and prime DNA.

What does clamp loader do?

Loads β clamp.
This keeps polymerase attached.

Difference between leading and lagging strand?

Leading is continuous.
Lagging is fragmented.

What is discontinuous replication?

Lagging strand synthesis in pieces.

What is an Okazaki fragment?

Short DNA segment on lagging strand.

What is the primer made of?

RNA.
DNA polymerase cannot start alone.

What removes the primer?

DNA Pol I.

Main replication enzyme?

DNA Pol III.

What connects fragments?

DNA ligase.

What is OriC?

Origin of replication in E. coli.

Why is AT-rich important?

A–T has weaker bonds.
It opens easily.

Why does DnaA need ATP?

ATP activates DnaA.
Only active DnaA can start replication.

How does DnaA initiate replication?

It binds oriC, melts DNA, forms open complex.

What does DnaC load?

DnaB helicase.

How many forks form?

2

 Why are TerA and TerB important?

They stop forks.
They ensure proper termination.

Why are TerA and TerB spaced apart?

To stop forks from both sides.

What does Tus do?

Binds ter sites and blocks helicase.

What does TUS stand for?

Terminus Utilization Substance.

Why is Topoisomerase IV important?

Separates linked chromosomes.

How can cells divide fast?

They start multiple rounds of replication.

Why is DNA supercoiled?

DNA is constrained in cells.
Twisting creates supercoils.

 Is DNA negatively or positively supercoiled? Why?

Negatively.
This counteracts positive stress.

How do Type I topoisomerases act?

Cut one strand, no ATP, relax DNA, +1 change.

 How do Type II topoisomerases act?

Cut two strands, use ATP, −2 change.

Type II enzyme that supercoils DNA?

Gyease

How were restriction enzymes discovered?

By observing phage restriction.

Role of restriction enzymes?

Destroy foreign DNA.

Why restriction/modification systems?

Modification protects self DNA.

What are orphan methylases?

Methylases without restriction enzymes.
Dam → adenine
Dcm → cytosine
They recognize specific sequences.

What is hemimethylated DNA and why is it important?

Only one strand is methylated.
This helps repair enzymes find the new strand.

Which class of restriction enzyme is commonly used in biotechnology laboratories in DNA cloning? Why this class and not the other classes?

Type II restriction enzymes are most commonly used in biotechnology laboratories.

They are used because:

1. Their restriction and methylation activities are in separate proteins, which makes them easier to control.

2. They cut DNA at the exact site where they bind, so scientists know exactly where the DNA will be cut.

Why is it important that restriction enzymes leave a 5′ phosphate and a 3′ hydroxyl group?

It is important because DNA ligase requires a 5′ phosphate and a 3′ hydroxyl to join DNA fragments.

If these groups were not present, ligase could not form phosphodiester bonds, and cloning would not work.

What are “sticky ends”? Why are they important in cloning experiments?

Sticky ends are single-stranded overhangs created when restriction enzymes cut DNA unevenly.

They are important because:

• They can base pair with complementary sticky ends

• This helps DNA fragments align correctly

• It increases the efficiency of ligation

Why is DNA ligase important for DNA cloning experiments?

DNA ligase joins DNA fragments together by sealing breaks in the backbone.

Without ligase, inserted DNA fragments would not become permanently attached to plasmids, so cloning would fail.

Why is gel electrophoresis important in restriction mapping experiments?

Gel electrophoresis separates DNA fragments by size.

This is important because:

• Scientists can see fragment lengths

• They can determine where restriction sites are

• They can verify if cutting was successful

What is Southern hybridization/blotting? Who invented it?

Southern hybridization is a method used to detect specific DNA sequences in a gel.

Steps:

• DNA is separated on a gel

• Transferred to a membrane

• Hybridized with a labeled probe

What is Northern hybridization/blotting?

Northern hybridization is similar to Southern blotting, but it detects RNA instead of DNA.

RNA is separated, transferred, and probed to study gene expression.

What is the principle behind Southern and Northern hybridization?

The principle is that complementary single-stranded nucleic acids will bind to each other.

A labeled probe finds and hybridizes to its matching sequence.

This allows specific sequences to be detected.

How is DNase I used in labeling DNA?

DNase I makes small nicks in the DNA backbone.

These nicks allow DNA polymerase to replace sections of DNA with labeled nucleotides during labeling.

What is the difference between nick translation and strand displacement synthesis?

• Nick translation:
  DNA Pol I removes nucleotides ahead of the nick and replaces them.

Strand displacement:
Klenow fragment synthesizes new DNA and displaces the old strand.

What is Klenow enzyme/fragment? What enzymatic properties does it have?

Klenow fragment is a part of DNA Polymerase I that lacks 5′→3′ exonuclease activity.

It has:

• 5′→3′ polymerase activity

• 3′→5′ proofreading activity

Can Klenow fragment do both nick translation and strand displacement?

No.
Klenow can only do strand displacement, not nick translation, because it lacks 5′→3′ exonuclease activity.

Can DNA Polymerase I do both nick translation and strand displacement?

Yes.
DNA Polymerase I can perform both, because it has exonuclease and polymerase activity.

In radioactive labeling, which phosphate must be radioactive?

The alpha phosphate must be radioactive.

This is because only the alpha phosphate becomes part of the DNA backbone.

What is the principle behind random priming labeling of DNA?

Random hexamer primers bind at many locations on DNA.

DNA polymerase extends from these primers, incorporating labeled nucleotides throughout the DNA.

This creates highly labeled probes.

What does PCR stand for?

PCR stands for Polymerase Chain Reaction.

Why is a thermostable DNA polymerase needed in PCR?

PCR requires repeated heating to separate DNA strands.

Normal enzymes would denature, but thermostable enzymes like Taq polymerase survive high temperatures.

Why are large molecules like biotin or digoxigenin used in nonradioactive labeling?

They are used because:

• They are easily detected

• They are safer than radioactive labels

• They can be recognized by antibodies

How are antibodies used in detection of labeled DNA?

Antibodies bind to biotin or digoxigenin.

These antibodies carry enzymes or fluorescent tags that produce detectable signals.

Difference between enzyme-mediated and fluorescence-mediated detection?

• Enzyme-mediated: Produces color or light using a substrate

• Fluorescence-mediated: Produces light directly

Who invented Sanger sequencing?

Frederick Sanger

100) What hydroxyl groups are missing in ddNTPs?

ddNTPs lack:

• 2′-OH

• 3′-OH