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General - Genetics 🧬 > L3: DNA Replication & Transcription > Flashcards

L3: DNA Replication & Transcription Flashcards

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1
Q

What is DNA replication?

A

Replication is:

  • the process by which an identical copy of DNA is made.
  • the duplication of the DNA content before mitosis i.e. the two DNA copies are distributed equally between the new daughter cells so that information can be preserved & given to offspring.
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2
Q

What are the general criteria for DNA replication?

A

β€œSTP 5 polymerase”

1) Replication is semiconservative (As each daughter DNA molecule contain):
 One old strand (one parent strand is conserved)
 One new strand (from free nucleotides in the nucleus)

2) Both strands serve as templates β€œsimultaneously”.
3) Replication is bidirectional.
4) Direction of replication: synthesis of new DNA in direction β€œ5β€² β†’ 3′”.
5) Done by DNA polymerases Complex

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3
Q

What are the components of DNA replication?

A

1) DNA template: Double-stranded DNA.

2) Precursors: dATP, dGTP, dCTP, dTTP.

3) Cofactors: Mg++, Mn++, ATP.

4) Enzymes and proteins: DNA polymerase, other enzymes, and proteins.

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4
Q

Characterises of DNA polymerases

A

1) Can read the template strand (3’ to 5’ direction) to synthesize a new strand in the 5’ to 3’ direction.

2) Deoxyribonucleotides triphosphate serve as precursors (dAMP, dGMP, dCMP and dTMP).

3) It cannot initiate DNA synthesis; it needs an RNA primer to build a new DNA strand upon it.

4) In eukaryotes, there are five types of DNA polymerases: Ξ±, Ξ΅, Ξ², Ξ³, and Ξ΄.

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5
Q

What are major enzymes needed for DNA replication?

A

DNA helicase
DNA primase
DNA polymerases
Exonucleases
Nick-sealing enzymes
SsDB Proteins

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6
Q

What is the function of DNA helicase?

A

Required for the unwinding of dsDNA.

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7
Q

What is the function of DNA primase?

A

❖ (a subunit of DNA polymerase Ξ±)
❖ Required for synthesis of RNA primer.

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8
Q

What is the function of DNA polymerases?

A

❖ required for deoxy-nucleotide polymerization and repair.

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9
Q

What is the function of exonucleases?

A

Remove RNA primers.

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10
Q

What is the function of neck-sealing enzymes?

A

DNA ligase

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11
Q

What are the steps of DNA replication?

A
  • Initiation or identification of ori
  • Unwinding and separation
  • Priming by primase
  • Synthesis of two complementary DNA strands
  • Excision of primers
  • Ligation of DNA fragments
  • Proofreading of newly synthesized DNA strands.
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12
Q

Initiation

A

❖ DNA replication starts at multiple sites (in Eukaryotes) called the origin of replication or (ori).

❖ These sites contain a short unique sequence of (rich in AT) base pairs (consensus sequence).

❖ DNA replication initiates at many different sites simultaneously and it is bidirectional.

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13
Q

Unwinding

A

❖ This origin of replication is unwound to generate a β€œreplication bubble” forming V-shaped two replication forks by DNA helicase enzyme (ATP-dependent).

❖ This process starts at each replication fork & proceeds in both directions.

❖ The two strands of DNA are kept away and separated by the single-strand binding proteins (ssBP).

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14
Q

Priming

A

❖ DNA polymerases cannot initiate DNA synthesis by themselves.

❖ The primase utilizes the DNA strands as templates and synthesizes a short stretch of RNA as a primer for DNA polymerase (a starting part used
in DNA replication).

❖ One RNA primer is formed for the leading strand and multiple primers for the lagging strand.

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15
Q

Synthesis

A

❖ The DNA polymerases are responsible for the synthesis of both strands of DNA.

❖ The presence of a large number of DNA polymerases (more than 20.000) helps to decrease the time needed for replication in eukaryotic cells.

❖ DNA polymerase reads the template strand (3’ to 5’ direction) to synthesize a new strand in the 5’ to 3’ direction.

❖ Deoxyribonucleotides triphosphate serve as precursors (dAMP, dGMP, dCMP and dTMP).

❖ The different bases are arranged in the complementary strands according to the sequence present in the parental strands.

❖ This can be completed by two different mechanisms on each strand: Leading and lagging

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16
Q

In what direction is the leading strand synthesized?

A

In the direction of the fork.

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17
Q

Is the leading strand continuous or not?

A

Yes

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18
Q

How many primers are there in the leading strand?

A

One

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19
Q

In what direction is the lagging strand?

A

Opposite to the direction of the fork.

20
Q

Is the lagging strand synthesized continuously or not?

A

No

21
Q

How many primers are found in the lagging strand?

A

Many

22
Q

Are the lagging strands formed from Okazaki fragments and what are they?

A

Yes and they are DNA + RNA primers

23
Q

Excision of RNA primers

A

❖ Removal of RNA primers by Exonulceases.

❖ Once the primers are removed, DNA polymerases land at the 3’ end of the preceding DNA fragment and extend the DNA over the gap.

24
Q

Ligation of DNA fragments

A

❖ The final phosphodiester linkage between the 5’-phosphate group on the DNA chain and the 3β€˜hydroxyl group on the adjacent chain is catalyzed by DNA ligase.

❖ This reaction requires ATP.

25
Q

Proofreading

A

❖ As each nucleotide is added to the chain, the DNA polymerases (Ξ΄ and Ξ΅) check the complementary base on the template.

❖ DNA polymerases hydrolytically remove the misplaced nucleotide (by the 3’ β†’ 5’ exonuclease activity) and replace it with the correct nucleotide.

26
Q

What are post replication modifications do DNA replication?

A

❖ The post-replication modifications of the newly synthesized DNA occur while being packaged into chromatin by the methylation of the 5th C atom of cytosine residues on DNA.

❖ Changing methylation status of DNA (Hypomethylation or Hypermethylation) is reported in different tumors.

27
Q

What are then inhibitors of DNA replication?

A

❖ Inhibitors of eukaryotic DNA replication are used as anticancer chemotherapy: Some act on DNA and some are nucleotides analogues

Act on DNA:❖ Actinomycin D:
 inhibit the initiation of replication by binding the DNA template.

Nucleotide analogues:❖ Cytosine arabinoside (ara C)
❖ 5-fluorouracil (thymine analogue).

28
Q

What is gene expression?

A

❖ The majority of genes are expressed as the proteins they encode.

❖ The process occurs in two steps :
 Transcription = DNA β†’ RNA
 Translation = RNA β†’ protein

29
Q

What is the structure of genes?

A

❖ Composed of exons, introns, and different control elements:
 Exon β†’ protein-coding sequence
 Intron β†’ intervening sequence (non-protein coding sequences)

30
Q

Transcription

A

1) Transcription, the synthesis of different types of RNA from a DNA template, is catalyzed by RNA polymerase.

2) DNA serves as the template for the synthesis of RNA much as it does for its own replication:
 The strand of DNA that is transcribed into RNA is called the template strand.
 The other strand of DNA is called the coding strand because it except for the change of U for T, its base sequence is identical to that of the RNA synthesized.

3) RNA is synthesized as a complementary strand from the DNA template strand, by the action of the enzyme RNA polymerase.

4) The basic mechanism of RNA synthesis is the same for all types of RNAs.

5) Eukaryotic RNA must travel from the nucleus to the cytoplasm for translation.

31
Q

What are the required materials for DNA transcription?

A

1) Template DNA: Transcription unit (promoter region, transcription start site, transcribed region, termination region, and regulatory element).

2) Four ribonucleotide triphosphate (ATP, GTP UTP, and CTP).

3) RNA polymerase enzymes

4) Transcription factors

32
Q

Eukaryotic RNA polymerase

A

❖ RNA polymerase read a DNA template in the 3‡ to 5‡ direction and synthesizes single-stranded RNA molecule in a 5‡ to 3‡ direction.

❖ It utilizes ribonucleoside triphosphates (NTP) as building units.

❖ Do not require a primer.

❖ Eukaryotes have several types of RNA polymerases.

❖ RNA polymerases have no known endonuclease or exonuclease activity. So, it has
no ability to repair mistakes in the RNA.

❖ There are three species of eukaryotic RNA polymerase: RNA polymerase I, RNA polymerase II, RNA polymerase III

33
Q

How is snRNA formed?

A

Mosy of it by RNA polymerase II but some with RNA polymerase III

34
Q

What are the steps of DNA transcription?

A

Initiation
Elongation
Termination

35
Q

Initiation of DNA transcription

A

❖ The binding of RNA polymerase to the promoter leads to local separation (unwinding) of the DNA double helix into template and coding strand.

❖ Several sequences are common to RNA polymerase II promoter and include:
 sequence formed of six nucleotides (TATAAA)
 The CAAT box (CCAATC) and the GC box (GGGCGG):

36
Q

TATAAA

A

β€’ located 25 to 35 base pairs upstream of the start point.

β€’ It binds several proteins termed TFIID which is the first step in transcription.

37
Q

CCAATC and GGGCGG

A

β€’ They determine the frequency of transcription events.

β€’ 70 to 80 base pairs upstream the start point.

β€’ Each of these boxes has its specific binding protein or proteins.

38
Q

Elongation of DNA transcription

A

❖ Sequential addition of ribonucleoside monophosphate.

❖ They are inserted in RNA molecules according to the bases pairing rule, PPI (pyrophosphate) is released (RNA is complementary to DNA template strand).
❖ Elongation of the RNA chain continues until a termination region is reached.

39
Q

Termination of DNA transcription

A

❖ Termination of transcription is started when RNA polymerase meets the terminator (termination signal is reached)

40
Q

What are post-transcriptional modifications?

A

❖ Primary mRNA is modified into mature mRNA in the nucleus by:
 Capping
 Tailing (addition of polyadenylate 3’ tail)
 Splicing (Removal of the intron).

41
Q

What is the definition of capping?

A

❖ It is the addition of 7 methyl guanosine triphosphate to the 5’ end of mRNA.

42
Q

What is the function of capping?

A

❖ protect the 5ΚΌ end of mRNA from attack by 5ΚΌ ribonucleases.

❖ it is important for initiation of translation through binding with the specific cap-binding protein

43
Q

What is tailing?

A

❖ Addition of up to 200 adenine nucleotides at the 3’ end by the action of poly-A- polymerase enzyme.

44
Q

What is the function of tailing?

A

❖ Protect the 3ΚΌ end mRNA from attack by 3ΚΌ ribonucleases

❖ It increases the efficiency of translation.

45
Q

How is splicing done?

A

❖ Done by several small nuclear RNA (snRNA)

46
Q

What happens to the remainder of the transcript?

A

Degraded.

47
Q

What are the inhibitors of DNA transcription?

A

❖ Act on DNA template: e.g. Actinomycin D

❖ Bind to RNA-polymerase: e.g. Ξ±- amanitin (eukaryotic RNA Polymerase II inhibitor).

General - Genetics 🧬 (8 decks)

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