Molecular Biology - 2.7 DNA Replication, Transcription & Translation

Question 1

Why is DNA replicated

Answer 1

DNA is replicated to produce an EXACT COPY of a CHROMOSOME in preparation for CELL DIVISION - when a cell divides during MITOSIS each new daughter cell must have a complete copy for the genetic code

this occurring during the “interphase of the mitosis cycles”

Question 2

Understandings:

Answer 2

1. the replication of DNA is semi-conservative and depends on complementary base pairing
2. Helicase (enzyme) unwinds the double helix and separates the two strands by breaking Hydrogen Bonds
3. DNA polymerase links nucleotides together to form a new strand, using the pre-existing strand as a template
4. Transcription is the synthesis of mRNA copied from the DNA base sequences by RNA polymerase

Question 3

Steps in DNA replication

Answer 3

1) Coiled DNA is allowed to uncoil
2) New pieves of DNA are formed

Question 4

1) Coiled DNA uncoils

Answer 4

The double helix is unwound by DNA GYRASE - an ENZYME called HELICASE breaks the weak hydrogen bonds between the bases - unzipping the rungs of the DNA ladder

Question 5

2) New pieces of DNA are formed

Answer 5

New pieces of DNA are formed from free nucleotide units joined together by the enzyme called DNA POLYMERASE (can only add bases in one direction (from the 5 prime to 3 prime end)

The free nucleotides are matched up to COMPLEMENTARY nucleotides in the original strand (A with T, G with C) = complementary strands

Question 6

OVERVIEW of gene expression (ie protein synthesis)

Answer 6

1. A section of DNA ( = a transcription unit) that codes for the polypeptide unzips
2. A copy of the transcription unit is made - mRNA (transcription)
3. mRNA moves out of the nucleus to ribosomes
4. three bases code for a specific amino acid
5. amino acids are assembled in the correct order by carrier tRNA to make the polypeptide chain (translation)
6. several polypeptides join together to make a protein

Question 7

Three bases that code for an amino acid - on DNA =

Answer 7

Triplets

Question 8

Three bases that code for an amino acid - on mRNA =

Answer 8

Codons

(“code for something”)

Question 9

Three bases that code for an amino acid - on tRNA =

Answer 9

Anticodons

(“complimentary to codon”)

Question 10

Role of codons (amino acids)

Answer 10

each triplet (group of 3 bases in DNA) codes for one amino acid - most amino acids have more than one triplet that codes for it
= the code = DEGENERATE (ie. flexibility, mutations in final base do not change amino acid made)

Many of the codons for a single amino acid differ ONLY IN THE LAST BASE (= reducing the chance that BASE MUTATIONS will have any NOTICABLE effect)

Question 11

“64 different combinations”

Answer 11

4 base codons (A,T,G,C) code for 64 different combinations = over 30 amino acids and the start/stop codons

Question 12

Refer to IRL notes for - DNA -> mRNA -> amino acids and how to find on table

Answer 12

Refer to IRL notes for - DNA -> mRNA -> amino acids and how to find on table

Question 13

Semi-Conservative

Answer 13

DNA = semi-conservative as: new double-stranded DNA molecule is formed and 1) one strand will be from the original template molecule, 2) one strand will be newly synthesised

• nitrogenous base can only pair with its complementary partner (ie A with T and C with 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 14

The theory that DNA replication was semi-conservative was confirmed by the:

Meselson-Stahl experiment in 1958

Answer 14

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

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

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

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 15

Helicase - DNA replication

Answer 15

Helicase unwinds the double helix and separates the two polynucleotide strands
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 16

DNA Polymerase - DNA replication

Answer 16

DNA polymerase synthesises new strands from the two parental template strands
Free deoxynucleoside triphosphates (nucleotides with 3 phosphate groups) align opposite their complementary base partner
DNA polymerase cleaves the two excess phosphates and uses the energy released to link the nucleotide to the new strand

Question 17

PCR - polymerase chain reaction

Answer 17

= 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 (230)

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

1. Denaturation – DNA sample is heated (~90ºC) to separate the two strands
2. Annealing – Sample is cooled (~55ºC) to allow primers to 3. anneal (primers designate sequence to be copied)
   Elongation – Sample is heated to the optimal temperature for a heat-tolerant polymerase (Taq) to function (~75ºC)

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 extends the nucleotide chain from the primers – therefore primers are used to select the sequence to be copied

Question 18

Transcription (SL - bioninja - more in 7.2)

Answer 18

Transcription is the process by which an RNA sequence is produced from a DNA template

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
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 19

Transcription / gene - continued

Answer 19

Gene =

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

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)

Transcription of genes occur in the nucleus (where DNA is), before the RNA moves to the cytoplasm (for translation)

Question 20

Codons

Answer 20

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 with a polypeptide chain
The order of the codons in an mRNA sequence determines the order of amino acids in a polypeptide chain

Question 21

Genetic Code

Answer 21

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 22

Translation (SL - bioninja - more in 7.2)

Answer 22

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

Ribosomes 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)
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

Messenger RNA (goes to…)
Ribosome (reads sequence in …)
Codons (recognised by …)
Anticodons (found on …)
Transfer RNA (which carries …)
Amino acids (which join via …)
Peptide bonds (to form …)
Polypeptides

Mnemonic: Mr Cat App

Question 23

Universality (application)

Answer 23

The genetic code is universal – almost every living organism uses the same code (there are a few rare and minor exceptions)

As the same codons code for the same amino acids in all living things, genetic information is transferrable between species

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)

Question 24

sequence decoding (skill!)

Answer 24

https://ib.bioninja.com.au/standard-level/topic-2-molecular-biology/27-dna-replication-transcri/sequence-decoding.html