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Mol Med > DNA and RNA > Flashcards

DNA and RNA Flashcards

1
Q

The building blocks of DNA are phosphate group, nitrogenous base and Pentose sugar.

What is the bond between phosphate group and pentose?

What is the bond between nitrogenous base and pentose (deoxyribose)?

A

Phosphodiester bond, phosphate group attached to 5’ on one nucleotide and with free hydroxyl group on the 3’ carbon of the next nucleotide

N-glycosidic bond

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2
Q

Separate them into pyrimidine and purine.

a. Adenine
b. Thymine
c. Guanine
d. cytosine
e. Uracil

A

Purine: pure as gold (adenine, guanine)

Others pyrimidine

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3
Q

Anticancer drugs like? intercalate into the minor groove – inhibit DNA and RNA synthesis

A

Anticancer drugs e.g actinomycin D (dactinomycin) intercalate into the minor groove – inhibit DNA and RNA synthesis

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4
Q

DNA replication is semi conservative. True or false?

A

True

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5
Q

In prokaryotes, RNA primers are removed by ? compared to

In eukaryotes, removed by ?

A

RNA primers are removed by DNA polymerase compared to RNase H and
FEN1 (Flap endonuclease 1) – removes RNA primer

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6
Q

Explain the difference between 3’ to 5’ exonuclease activity and 5’ to 3’ exonuclease activity?

A

1, DNA polymerase reverse its direction if base pairs mismatched.

Direction 3’ to 5’

  1. Removes ribonucleotide e.g RNA primer sequences of Okazakifragments or dNTPS at 5’ or some units away from the 5’ end.

Direction 5’ to 3’

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7
Q

The reaction used to obtain energy from to catalyst strand separation and formation of replication fork?

A

Nucleotide hydrolysis

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8
Q

DNA primase in prokaryote and eukaryote

A

DNA pol I (prokaryote) and DNA pol α (eukaryote)

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9
Q

1) Initiation steps in DNA synthesis

  1. —-protein binds to specific repeat
    sequences of 9 nucleotides in OriC
  2. Interaction of —- protein induces
    unwinding at the AT rich region
    Why choose AT rich region?
  • opening of double helix strands and formation of replication forks
  1. Binding of —- and —— complex contain helicase
    proteins further breaks H-bonds ahead of the replication forks
  2. —-keep the two strands apart
  3. —- binding completes the
    primosome
A

OriC is recognised by a pre-priming complex of proteins

DnaA, DnaA, DnaB, DnaC, Single stranded binding protein (SSB), DnaG

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10
Q

End replication problem. At the extreme end of a chromosome, there is no way to synthesize this region when the last primer is removed. Hence, cause what?

A

The lagging strand is always shorter than its template by at
least the length of the primer. • Somatic cells: Telomere shortening beyond critical length →
senescent (aging)

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11
Q

The PCR requires three processes: denaturation, annealing and extension. Explain (the enzymes involved are DNA primers and Taq polymerase. Is Taq polymerase heat tolerant.

A

Denaturation: DNA sample is heated to DNA sample is heated to separate it into two single strands (~95ºC for 1 min)

Annealing: DNA primers attach to the 3’ ends of the target sequence (~55ºC for 1 min)

Extension: A heat-tolerant DNA polymerase (Taq) binds to the primer and copies the strand (~72ºC for 2 min). The new strands extend from 5’ to 3’ direction
Yes.

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12
Q

Why is melting temperature ( to cause DNA half denatured ) is dependent on the length and composition of the bases (G-C content)?

Other conditions where DNA denatured? (2)

A

Stronger bond in GC requires more heat energy to break the bond.

Extrem pH (abundant OH- ion)

Low salt concentrations

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13
Q

• The total amount of pyrimidines (T+C) always equals the
total amount of purines (A+G) • The amount of T always equals the amount of A, and the
amount of C always equal the amount of G. But the amount
of A+T is not necessarily equal to the amount of G+C.

Name the rule.

A

Chargaff’s Rule

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14
Q

Only 25000 encoding genes in human. Non-coding DNA in the human genome contains repeat sequences: (a) tandem repeat (b) interspersed repeat.

  1. —- are two or more nucleotides repeated as a unit one after another
    in the same orientation. (Contain —- satellites and —— satellites)
  2. also known as retrotransposons, are
    repeat elements characterized by RNA intermediates (constitutes ~50% genome)

(a) LT R (long terminal repeat) retrotransposons

b) non-LTR retrotransposons.
e.g. —-
—-

A

Tandem repeats

Interspersed repetitive DNA
non-LTR retrotransposons. - SINEs (short interspersed nuclear elements, <500bp) 14% - LINEs (long interspersed nuclear elements) 20%

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15
Q

There are different types of RNA. Fill in these RNA according to their functions?

  1. ——provides link from gene to protein
  2. —— ribosome structure/function; protein synthesis
  3. —— recruit amino acids for protein synthesis

Other
1. ——processing of mRNA
2. ——regulation of gene expression
3. ——inactivation of one X chromosome (females)
4. ——RNAs with catalytic activity

A
  1. Messenger RNA (mRNA)
  2. Ribosomal RNA (rRNA)
  3. Transfer RNA (tRNA)

Small nuclear RNAs
Micro RNA
XIST RNA
Ribozymes

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16
Q

Why is X inactivation needed? What is the RNA involved?

A

Ensure that female has only one functional copy of X chromosome in each somatic cells except for germ cells (like male)

*happens in early embryonic development in females

XIST RNA

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17
Q

These structural genes undergo transcription and translation to produce these proteins. Form what protein?

  1. lacZ
  2. lacY
  3. lacA
A

Beta-galactosidase
Permease
Transacetylase

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18
Q
  1. Base + sugar + phosphate
  2. Base + sugar
A
  1. Nucleotide
  2. Nucleoside
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19
Q

We have 5 DNA polymerase in eukaryotic cells (alpha, beta, delta, epsilon, gamma. State the function and direction for exonuclease activity.

A
  1. Alpha: synthesize RNA primer for lagging strand
  2. Beta: repairs DNA
  3. Delta: remove primer + fill in gaps
  4. Epsilon:synthesize leading strand
  5. Gamma: replicates mitochondrial DNA
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20
Q

Which DNA polymerase is responsible to add nucleotides?

A

DNA polymerase III

21
Q

Mechanism of telomere elongation by telomerase

A

Telomerase: a complex that carries a protein (TERT) and internal RNA sequences that act as a template

Telomerase extends the 3’ end (the longer strand) of the DNA using the internal RNA sequences as a template
The extended DNA now becomes a template for normal DNA synthesis

22
Q

All RNA in prokaryotes are produced by?
For eukaryotes, different RNAs require different types of polymerase
1. rRNA 28S, 18S & 5.8S
2. mRNA, snRNAs & miRNAs
3. rRNA 5S; all tRNAs

A

RNA polymerase I (Pol I): rRNA 28S, 18S & 5.8S
RNA polymerase II (Pol II): mRNA, snRNAs & miRNAs
RNA polymerase III (Pol III): rRNA 5S; all tRNAs

23
Q

In eukaryotes, how are RNA molecules processed after transcription?

A

they are spliced and have a 5’ cap and poly-A tail
put on their ends.

24
Q

How is core enzyme different from holoenzyme? State the function of all subunits in RNA polymerase?

A

Sigma unit is added to core enzyme to form holoenzyme.
* different sigma factor will recognize different genes s70 s 28 s 32 s 54
Alpha: bind to regulatory region
Beta: forms phosphodiester bond
Beta ‘ :bind to template
Sigma: recognise promoter region

25
Q

Do transcription by RNA polymerase requires primer?

A

No

26
Q

Explain initiation stage of transcpription.

A

I. Promoter recognition
II. Formation of transcription
bubble
III. Formation of first bond

between ribonucleoside tri-
phosphate(rNTPs)

Details:
1. The sigma factor associates with the core enzyme to form a holoenzyme which binds to the -35 and -10 consensus sequences in the promoter, creating a closed complex.

  1. The holoenzyme binds to the promoter tightly and unwinds the double-stranded DNA, creating an open complex.
  2. A nucleoside triphosphate complementary to the DNA at the start site serves as the first nucleotide in the RNA molecule.
  3. Two phosphate groups are cleaved from each subsequent nucleoside triophosphate, creating an RNA nucleotide that is added to the 3’ end of the growing RNA molecule. (meaning synthesis is 5’ to 3’)
  4. The sigma factor is released as the RNA polymerase move beyond the promoter.
27
Q

What are the two Prokaryotic promoter: contains consensus sequences (short stretch of common nucleotides) which is used for contact of holoenzyme and creates a transciption bubble.

A
  • -35 sequence (5TTGACA-3)
  • Pribnow box (5-TATAAT-3)
28
Q

RNA polymerase is a holoenzyme where core enzyme + ? Will recognise DNA region.

A

Sigma factor

29
Q

During elongation phase of transcription, what is the main events that happened?

A

RNA pol uses nucleoside triphosphate and releases
pyrophosphate each time a nucleoside monophosphate is added to the chain.

DNA-RNA hybrid helix is formed.

30
Q

Do RNA polymerase have proofreading?

A

No

31
Q

RNA synthesis continues along the DNA template until RNA pol encounters specific sequences on DNA called?

A

Terminators

32
Q

There are two types of terminations. Explain the difference.
1. rho independent termination
2. rho dependent termination

A

ρ (rho) dependent termination
* Requires protein ρ (rho).
* Rho binds to “rho recognition site” near 3` end of new RNA.
* Moves along RNA until it reaches RNA pol paused at termination
site.
* ATP-dependent helicase activity of rho separates RNA-DNA hybrid.

ρ (rho) independent termination
* depends on specific sequences on the DNA strand
* When the polymerase approaches the end of a gene, it encounters
a region rich in G-C nucleotides.

  • The mRNA then folds back on itself, allowing the complementary G-
    C nucleotides to bind, forming a stable hairpin structure.
  • It causes the polymerase to stall transcription of a region rich in A–T
    nucleotides.
33
Q

The coding regoin of the eukaryotic gene is?

A

Exon.
Intron is the non-coding region, removed during mRNA processing

34
Q

Why is transcription factor important?

A

TFs are required for assembly of transcription complex at promoter and determination of which genes are to be transcribed

35
Q

Explain the functions of these proteins in transcription.
1. Basal transcription factors
2. Promoter
3. Coactivators
4. Activators
5. Repressors

A
  1. positions RNA polymerase at the start of the protein-coding region of the gene and send the enzyme on its way
  2. Regulates gene transcription
  3. Integrate signals from activators and repressors (relay the results of basal factors)
  4. Activators: bind to enhancers in genes (in upstream regulatory sequence). Speed up transcription.
  5. Repressors: bind to silencers. Slow down transcription.
    TATA box is a DNA sequence that indicates where a genetic sequence can be read and decoded. It is a type of promoter sequence, which specifies to other molecules where transcription begins.
36
Q

What is the 5’ end of mRNA capped by? And what are the functions?

A

Methy guanosine residue

  1. Protects from degradation (by 5’ exonuclease) during elongation of RNA
    chain
  2. Help the transcript bind to the ribosome during protein synthesis
37
Q

Polyadenylation signal near 3’ end - what sequence? for the recognition by specific endonuclease that cleaves RNA

Why is the enzyme involved?

Why is poly (A) tail important?

A

AAUAAA consensus sequence

Poly (A) polymerase

Makes the transcript more stable and helps it get exported from
the nucleus to the cytosol.

38
Q

Explain splicing in post-transcriptional processing?

A

Introns are removed by spliceosome and exons are spliced together to form mature mRNA. Later be transported into cytoplasm through the nuclear membrane.

39
Q

List one mRNA that undergoes alternative splicing.

A

mRNA of Tropomyosin (actin filament binding
protein) undergoes tissue-specific alternative splicing and
produces multiple isoforms of tropomyosin protein

40
Q

pre-rRNAs are cleaved by?

A

Ribonuclease. rRNA processing occurs in nucleolus

41
Q

This question is regarding pre tRNA to Mature tRNA. 4 major steps
1. 16- nucleotide at the 5’ end is cleaved by?
2. 14-nucleotide in the anticodon loop is removed by?
3. Uracil residues at the 3’ end are replaced by?
4. Many bases are converted to characteristic modified bases.

A
  1. RNase P
  2. nucleases
  3. CCA sequence
42
Q

Three inhibitors for transcription.
1. Rifampin
2. Dactinomycin *(actonomycin D)
3. alpha- amanitin

A
  1. -inhibits initiation of transcription by binding to β subunit of RNA pol
    -Used to treat tuberculosis
    *prokaryotic
    • Intercalates (binds) to DNA template and
      interferes with movement of RNA pol.
      * Inhibits prokaryotic and eukaryotic
      transcription
      * Anti-cancer activity – e.g Wilm’s tumour,
      rhabdomyosarcoma, and Ewing’s sarcoma
    • Tightly binds to eukaryotic RNA polymerase II and disrupts the elongation phase
      of transcription
      * Blocks translocation of the transcription complex in the elongation step
      * Produced by poisonous mushroom Amanita phalloides
43
Q

Explain the difference between positive sense RNA and negative sense RNA?

A

Positive Sense RNA: recognized as mRNA; immediately translated
by host cell

Negative Sense RNA: template for viral RNA-directed
mRNA synthesis

44
Q

Some examples of pathogenic RNA
viruses
1. ds RNA:

  1. Positive sense ss RNA:
  2. Negative sense ss RNA:
A
  1. Rotavirus
  2. SARS, Hepatitis C, Retroviruses*
  3. Influenza, Measles, Mumps,
    Rabies
45
Q

Explain retroviruses?

A
  • Positive sense ss RNA
  • 5’-cap (for ribosomal recognition)
  • poly A tail (promote stability)
  • Viral reverse transcriptase
  • Integrated in to cell genome via integrase
46
Q

There are a few steps in reverse transcription of retroviruses.

A
  1. Endocytosis
  2. Uncoating
  3. Viral DNA –> DNA-RNA Hybrid –> ss DNA –> ds DNA (reverse transcription is from RNA to DNA)
  4. Proviral DNA is integrated into genome by integrase
  5. Undergo transcription to form mRNA
  6. Viral RNA exits out into cytoplasm through nuclear pores
  7. Assembly of viral DNA
  8. Budding out of cell
47
Q

Explain RNA directed DNA synthesis.

A
  1. Virus corrupts tRNA as primer for RNA-directed DNA synthesis
  2. Viral reverse transcriptase synthesises terminal segment of cDNA
  3. Primer jumps to 5’ end of viral RNA; full length first strand cDNA synthesised
48
Q

Explain reverse transcription PCR.

A
  1. Oligodt Primer binds to Poly (A)
    tail
  2. mRNA-DNA hybrid formed (reverse transcriptase + dNTP)
  3. mRNA is degraded with alkali
  4. New single-stranded cDNA is the one left for normal transcription
  5. Now become duplex DNA by DNA polymerase I (PCR) and dNTP
  6. Now can undergo a lot of cDNA
49
Q
A

Mol Med (3 decks)

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