Molecular Biology - 2.7 DNA Replication, Transcription & Translation Flashcards
Why is DNA replicated
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”
Understandings:
- the replication of DNA is semi-conservative and depends on complementary base pairing
- Helicase (enzyme) unwinds the double helix and separates the two strands by breaking Hydrogen Bonds
- DNA polymerase links nucleotides together to form a new strand, using the pre-existing strand as a template
- Transcription is the synthesis of mRNA copied from the DNA base sequences by RNA polymerase
Steps in DNA replication
1) Coiled DNA is allowed to uncoil
2) New pieves of DNA are formed
1) Coiled DNA uncoils
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
2) New pieces of DNA are formed
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
OVERVIEW of gene expression (ie protein synthesis)
- A section of DNA ( = a transcription unit) that codes for the polypeptide unzips
- A copy of the transcription unit is made - mRNA (transcription)
- mRNA moves out of the nucleus to ribosomes
- three bases code for a specific amino acid
- amino acids are assembled in the correct order by carrier tRNA to make the polypeptide chain (translation)
- several polypeptides join together to make a protein
Three bases that code for an amino acid - on DNA =
Triplets
Three bases that code for an amino acid - on mRNA =
Codons
(“code for something”)
Three bases that code for an amino acid - on tRNA =
Anticodons
(“complimentary to codon”)
Role of codons (amino acids)
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)
“64 different combinations”
4 base codons (A,T,G,C) code for 64 different combinations = over 30 amino acids and the start/stop codons
Refer to IRL notes for - DNA -> mRNA -> amino acids and how to find on table
Refer to IRL notes for - DNA -> mRNA -> amino acids and how to find on table
Semi-Conservative
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
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:
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
Helicase - DNA replication
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
DNA Polymerase - DNA replication
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
PCR - polymerase chain reaction
= 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:
- Denaturation – DNA sample is heated (~90ºC) to separate the two strands
- 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
Transcription (SL - bioninja - more in 7.2)
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
Transcription / gene - continued
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)
Codons
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
Genetic Code
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
Translation (SL - bioninja - more in 7.2)
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
Universality (application)
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)
sequence decoding (skill!)
https://ib.bioninja.com.au/standard-level/topic-2-molecular-biology/27-dna-replication-transcri/sequence-decoding.html
