Molecular Biology: 2.7 DNA replication, transcription, and translation

Question 1

How is DNA a semi-conservative process?

Answer 1

DNA replication is a semi-conservative process, because when a new double-stranded DNA molecule is formed:

One strand will be from the original template molecule
One strand will be newly synthesised

Question 2

What causes DNA to replicate in a semi-conservative process and what is the consequence?

Answer 2

This occurs because each nitrogenous base can only pair with its complementary partner

Adenine (A) pairs with thymine (T)
Cytosine (C) pairs with guanine (G)

Consequently, when DNA is replicated by the combined action of helicase and DNA polymerase:

Each new strand formed will be identical to the original strand separated from the template
The two semi-conservative molecules formed will have an identical base sequence to the original molecule

Question 3

The theory that DNA replication was semi-conservative was confirmed by the Meselson-Stahl experiment in 1958

Prior to this experiment, three hypotheses had been proposed for the method of replication of DNA:

Answer 3

Conservative Model – An entirely new molecule is synthesised from a DNA template (which remains unaltered)
Semi-Conservative Model – Each new molecule consists of one newly synthesised strand and one template strand
Dispersive Model – New molecules are made of segments of new and old DNA

Question 4

What was the Meselson and Stahl experiment?

Answer 4

Meselson and Stahl were able to experimentally test the validity of these three models using radioactive isotopes of nitrogen

Nitrogen is a key component of DNA and can exist as a heavier 15N or a lighter 14N

DNA molecules were prepared using the heavier 15N and then induced to replicate in the presence of the lighter 14N

DNA samples were then separated via centrifugation to determine the composition of DNA in the replicated molecules

Question 5

What were the results of the Meselson and Stahl experiment?

Answer 5

The results after two divisions supported the semi-conservative model of DNA replication

After one division, DNA molecules were found to contain a mix of 15N and 14N, disproving the conservative model
After two divisions, some molecules of DNA were found to consist solely of 14N, disproving the dispersive model

Question 6

The process of DNA replication is coordinated by two key enzymes – helicase and DNA polymerase. Explain the function of helicase

Answer 6

Helicase unwinds the double helix and separates the two polynucleotide strands, resulting in a replication fork
It does this by breaking the hydrogen bonds that exist between complementary base pairs
The two separated polynucleotide strands will act as templates for the synthesis of new complementary strands

Question 7

Explain the function of DNA polymerase

Answer 7

DNA polymerase synthesises new strands from the two parental template strands

Free deoxynucleoside triphosphates (nucleotides with 3 phosphate groups found floating freely in the nucleoplasm,) align opposite their complementary base partner (and forms hydrogen bonds)

DNA polymerase cleaves the two excess phosphates and uses the energy released to link the nucleotide to the new strand by making covalent bonds between the phosphate group of the free nucleotide and the sugar of the adjoining nucleotide on the new strand.

Question 8

Outline the direction in which DNA polymerase works

Answer 8

DNA polymerase links free nucleotides in only one direction that is 5’ to 3’ (leading strand). This is because the pentose sugar is the 3’ terminal and the phosphate group is the 5’ terminal and DNA polymerase links the phosphate group to the pentose sugar.

The other new strand being formed, the lagging strand is synthesised in the opposite direction (3’ to 5’)

Question 9

What is the polymerase chain reaction and what is it used for?

Answer 9

The polymerase chain reaction (PCR) is an artificial method of replicating DNA under laboratory conditions

The PCR technique is used to amplify large quantities of a specific sequence of DNA from an initial minute sample
Each reaction doubles the amount of DNA – a standard PCR sequence of 30 cycles creates over 1 billion copies

Question 10

Explain the process of polymerase chain reaction

Answer 10

The reaction occurs in a thermal cycler and uses variations in temperature to control the replication process via three steps:

Denaturation – DNA sample is heated (~90ºC) to separate the two strands
Annealing – Sample is cooled (~55ºC) to allow primers to anneal (primers designate sequence to be copied). Because they are present in excess, they prevent the re-annealing of the parent strands.
Elongation – Sample is heated to the optimal temperature for a heat-tolerant polymerase (Taq) to function (~75ºC)

Question 11

Explain the nature and role of Taq in polymerase chain reaction

Answer 11

Taq polymerase is an enzyme isolated from the thermophilic bacterium Thermus aquaticus

As this enzyme’s optimal temperature is ~75ºC, it is able to function at the high temperatures used in PCR without denaturing
Taq polymerase synthesises double stranded DNA at a very rapid rate using single strands with primers as templates – therefore primers are used to select the sequence to be copied

Question 12

Describe the process of transcription

Answer 12

Transcription is the process by which an RNA sequence is produced from a DNA template. Transcription of genes occur in the nucleus (where DNA is), before the RNA moves to the cytoplasm (for translation)

RNA polymerase separates the DNA strands and synthesises a complementary RNA copy from one of the DNA strands
When the DNA strands are separated, ribonucleoside triphosphates align opposite their exposed complementary base partner (uracil pairs in complementary fashion with adenine)
RNA polymerase removes the additional phosphate groups and uses the energy from this cleavage to covalently join the nucleotide to the growing sequence
Once the RNA sequence has been synthesised, RNA polymerase detaches from the DNA molecule and the double helix reforms

Question 13

What is a gene in terms of transcription?

Answer 13

The sequence of DNA that is transcribed into RNA is called a gene

Question 14

What is antisense and sense in transcription?

Answer 14

The strand that is transcribed is called the antisense strand and is complementary to the RNA sequence
The strand that is not transcribed is called the sense strand and is identical to the RNA sequence (with T instead of U)

Question 15

Explain what are codons?

Answer 15

The base sequence of an mRNA molecule encodes the production of a polypeptide
The mRNA sequence is read by the ribosome in triplets of bases called codons
Each codon codes for one amino acid within a polypeptide chain
The order of the codons in an mRNA sequence determines the order of amino acids in a polypeptide chain

Question 16

What is genetic code?

Answer 16

The genetic code is the set of rules by which information encoded within mRNA sequences is converted into amino acid sequences (polypeptides) by living cells
The genetic code identifies the corresponding amino acid for each codon combination
As there are four possible bases in a nucleotide sequence, and three bases per codon, there are 64 codon possibilities (43)
The coding region of an mRNA sequence always begins with a START codon (AUG) and terminates with a STOP codon

Question 17

What is anticodon and its function?

Answer 17

An anticodon is a sequence of three transfer RNA (tRNA) bases that are complementary to a codon
The transfer RNA carries the appropriate amino acid to the ribosome
The amino acid can then be condensed onto the growing polypeptide chain

Question 18

Define translation

Answer 18

Translation is the process of protein synthesis in which the genetic information encoded in mRNA is translated into a sequence of amino acids on a polypeptide chain

Question 19

Outline the process of translation

Answer 19

Ribosome small subunit bind to mRNA in the cytoplasm and move along the molecule in a 5’ – 3’ direction until it reaches a start codon (AUG)
Anticodons on tRNA molecules align opposite appropriate codons according to complementary base pairing (e.g. AUG = UAC)
Each tRNA molecule carries a specific amino acid (according to the genetic code) and accommodates in the large subunit (2 tRNAs can fit at a time)
Ribosomes catalyse the formation of peptide bonds between adjacent amino acids (via condensation reactions)
The ribosome moves along the mRNA molecule synthesising a polypeptide chain until it reaches a stop codon
At this point translation ceases and the polypeptide chain is released

Question 20

Outline production of human insulin from bacteria

Answer 20

The genetic code is universal – almost every living organism uses the same code

The ability to transfer genes between species has been utilised to produce human insulin in bacteria (for mass production)

The gene responsible for insulin production is extracted from a human cell
It is spliced into a plasmid vector (for autonomous replication and expression) before being inserted into a bacterial cell
The transgenic bacteria (typically E. coli) are then selected and cultured in a fermentation tank (to increase bacterial numbers)
The bacteria now produce human insulin, which is harvested, purified and packaged for human use (i.e. by diabetics)