2.7 dna replication, transcription, translation Flashcards
1
Q
what model does dna replication follow? (conservative, semiconservative, dispersive etc)
A
- dna replication follows the semi-conservative model
- involves separation of DNA of the parental
double-stranded DNA - each of this separated strands are used a template to synthesise the complimentary strand
- each of the resultant replicated double stranded DNA will consist of one strand from the original DNA double-strand while the other strand is newly synthesised
2
Q
what is helicase?
A
- enzyme essential for DNA replication
- unwinds the double helix of the double-stranded DNA
- accomplished by breaking the hydrogen bonds between complimentary base pairs holding the double strands together
3
Q
what is a replication bubble?
A
- dna replication begins at the origin with helicase unwinding the DNA
- creates something known as a “replication fork”
- as replication takes place at both directions at the same time, two replications working together will form something known as a replication bubble
4
Q
what is dna polymerase?
A
- class of different enzymes that are responsible for the synthesis of new DNA strands
- accomplished when the enzyme facilitates two things:
• entry (or in a certain way the selection) of a complimentary nucleotide based on the DNA template (complimentary strand)
• catalysis of a phosphodiester bond between this new nucleotide with the last nucleotide (3’ end) on the existing newly synthesised strand
5
Q
how does dna polymerase operate?
A
- always moves in a 5’ to 3’ direction (new strand)
- new nucleotide can only be added by polymerase on the 3’ end of the existing newly synthesised strand
- able to catalyse the phosphodiester bond between adjacent nucleotides, thus lengthening the strand
- proof reading function is built in the enzyme, and when the wrong nucleotide (base) is added to the strand, the
entire enzyme will pause and carry processes to remove the wrong nucleotide before redoing that portion - uses free floating nucleotides in the cytoplasm as raw material to synthesise the new strand
• nucleotides exists as dATP, dTTP, dGTP and dCTP
• (deoxy-adenine triphosphate, deoxy-thymine triphosphate etc)
• presence of the additional phosphate groups allows for the supply of chemical energy to drive the synthesis process
6
Q
how did meselson and stahl obtain support for the theory of semi-conservative replication of dna?
A
- initially, 3 models proposed to investigate how DNA is replicated and inherited by the daughter cells after cell division:
• conservative
• semi-conservative
• dispersive - meselson and stahl conducted experiments to prove that DNA is inherited in a semi- conservative manner
- first cultured E. coli in media with nutrients that contain only 15N nitrogen, for many generations
- 15N nitrogen is an isotope of nitrogen, and the the common form of nitrogen nature is 14N nitrogen
- bacterial cells were then transferred to media that contain only 14N nitrogen, and are allowed to grow
- samples of bacterial cells were removed for each subsequent generation of cells (generation time of E coli is around 20 minutes)
- samples were tested to determine the types of nitrogen isotopes present in the DNA
- to analyse the different types of DNA present in the bacterial cells, a salt density gradient was used
- tube containing layers of different concentrations of Caesium chloride is prepared
- when DNA is placed at the top of the tube and centrifuged, the DNA will separate based on different densities
- DNA in naturally occurring cells are of the same density as they are made using N14 (normal atmospheric nitrogen)
- DNA made using an isotope of nitrogen 15N will be denser than normal DNA made from 14N
- when centrifuged, DNA made from N15 will sink lower than DNA made from N14 when they are placed in a Caesium chloride density gradient solution
- DNA can be radioactively labeled and detected using photographic film
7
Q
why is Taq DNA polymerase used in pcr?
A
- pcr is effective method to greatly amplify a portion of DNA
- requires the use of Taq polymerase, as the temperatures involved at much higher than those normal terrestrial organisms (and hence enzymes) are exposed to
• taq is short for thermophilus aquaticus - high temperatures involved allows for rapid separation and replication of DNA strands without sacrificing accuracy of replication (as opposed to running PCR at room temperatures)
8
Q
how are primers involved in pcr?
A
- pcr involves use of primers
- primers: short single stranded sequences of DNA, and
the use of primers means that primase is not required - 2 primers needed, a forward primer and a reverse primer, and these two primers are meant to be used on the two different strands on a double stranded piece of DNA
- region that is amplified by pcr will be the region between the two primers (including the sequences on the primers)
- hence, these primers are supposed to be determined prior to the PCR, and are chemically synthesised (engineered) specifically for the experiment (unique)
9
Q
what are the steps in pcr?
A
- involves repeated cycles of three steps, and at the end of each cycle the amount of DNA is doubled
• 1 cycle of PCR yields two identical copies of the DNA sequence
• standard reaction of 30 cycles would yield 1,073,741,826 copies of DNA (2^30)
3 steps are:
- denaturation – the separation of double stranded DNA into single strands (using heat)
- annealing – the two sets of primers attach on opposite ends of the target sequence, and the two sets bind onto different strands
- extension – heat tolerant Taq polymerase synthesises the complimentary strands by extending the primers in the 3’ direction
10
Q
what are the advantages of pcr?
A
- can amplify very small amounts of DNA (especially useful in forensic work) - highly specific and will amplify a specific region of DNA as determined by the two primers used - able to give good results rapidly • each cycle of PCR involves: • 15 seconds of denaturation • 15 seconds for primer annealing • 45 seconds for extension
11
Q
what is transcription?
A
- the synthesis of mRNA copied from the DNA base sequences by RNA polymerase
- genetic information stored in form of DNA are mainly information that meant for production of specific proteins in cells
- involves transcription of DNA into mRNA, and mobile mRNA moves to ribosomes where translation
occurs to translate the information in genetic code (A,G, C and T) into sequences of amino acids in polypeptide chains
12
Q
what are the 3 phases of transcription?
A
- initiation phase: where the RNA polymerase attaches to the promoter sequence upstream from the gene sequence
- elongation phase: where the RNA polymerase helps to facilitate the binding of a complimentary RNA nucleotide to the nucleotide on the template DNA, and also in the catalysis of the formation of the phosphodiester bond between the adjacent RNA nucleotides
• as RNA grows longer, the mRNA peels away and the DNA strand immediately forms back the double helix structure with the other strand - termination phase: where specific sequences on the DNA template (terminator sequences) signal the detachment of the RNA polymerase and the mRNA
13
Q
what is translation?
A
- synthesis of polypeptides on ribosomes
- translation of mRNA is essential in the formation of new polypeptide strands in the cell
- genetic information existing as triplet codons on the mRNA used to determine the corresponding arrangement of amino acids in the new polypeptide molecule
- ribosomes are the main molecules that carry out the translation process
- also involves tRNA molecules that are bonded to specific amino acid molecules, besides the mRNA strand
14
Q
what is the purpose of ribosomes in translation?
A
- coordinate the functioning of mRNA and tRNA and, ultimately, the synthesis of polypeptides
- have 2 subunits: small and large
- each subunit is composed of ribosomal RNAs and
proteins - come together during translation
- ribosomes have binding sites for mRNA and tRNA molecules
- can be found as free floating ribosomes, and in most eukaryotic cells, ribosomes can also be found bound on the surface of the rough endoplasmic reticulum
15
Q
what is genetic code?
A
- system that determines which sequences of nucleotides on the mRNA codes for which amino acid
- universal, and is the same in almost all species of life on earth
- same piece of mRNA produced by one species of cell can be used by the cell of another species to produce the same polypeptide chain
- amino acid sequence of polypeptides is determined by mRNA according to the genetic code
