Topic 7 Flashcards
Describe the Hersey and Chase experiment
- provided evidence that DNA is the genetic material
- T2 bacteriophage were grown in two isotopic mediums
- viruses were grown in radioactive sulfur with radiolabelled proteins as sulfur is present in proteins but not DNA
- and viruses were also grown in radioactive phosphorous with radiolabelled DNA ( phosphorous is present in DNA)
- the viruses then infected bacteria and the two were separated by centrifugation
- The bacteria was found to be radioactive when infected by the 32P–viruses (DNA) but not the 35S–viruses (protein)
Describe x ray diffraction
- Used to investigate the structure of DNA
- DNA was purified and then fibres were stretched in a thin glass tube
- The DNA was targeted by a X-ray beam, which was diffracted when it contacted an atom
- The scattering pattern of the X-ray was recorded
What evidence did xray diffraction provide for the structure of DNA?
- double helix structure
- double stranded
How does the structure of DNA suggest a mechanism for DNA replication?
- Chargaff had also demonstrated that DNA is composed of an equal number of purines (A + G) and pyrimidines (C + T) .Shows that nitrogenous bases are paired within the double helix and the two strands must run in anti-parallel directions
- Franklin’s x-ray diffraction experiments demonstrated that the DNA helix with P on the outside. The P form an outer backbone and nitrogenous bases are packaged within the interior
- DNA replication occurs by complimentary base pairing and is bidirectional
Describe the process of DNA replication
- Helicase unwinds and separates the double-stranded DNA by breaking the hydrogen bonds between base pairs creating a replication fork of two strands running in antiparallel directions
- DNA gyrase relieves the tension in the strand.
- DNA primase generates a short RNA primer providing an initiation point for DNA polymerase III, which can extend a nucleotide chain but not start one.
- Free nucleotides align opposite their complementary base partners and DNA pol III attaches to the 3’-end of the primer and covalently joins the free nucleotides together in a 5’ → 3’ direction
As DNA strands are antiparallel, DNA pol III moves in opposite directions on the two strands
On the leading strand, DNA pol III is moving towards the replication fork and can synthesise continuously
On the lagging strand, DNA pol III is moving away from the replication fork and synthesises in pieces (Okazaki fragments). - As the lagging strand is synthesised in a series of short fragments, it has multiple RNA primers along its length
DNA pol I removes the RNA primers from the lagging strand and replaces them with DNA nucleotides - DNA ligase joins the Okazaki fragments together to form a continuous strand by covalently joining the sugar-phosphate backbones together with a phosphodiester bond
What can you use to stop DNA replication in preparation of samples for base sequencing?
dideoxyribonucleic acid
What is DNA sequencing?
Making the base order of a nucleotide sequences clearer
What about dideoxynucleotides make them stop DNA replication?
-Dideoxynucleotides (ddNTPs) lack the 3’-hydroxyl group necessary for forming a phosphodiester bond which prevent further elongation of a nucleotide chain and terminate replication.
Describe Sanger’s method for DNA sequencing
- Four PCR mixes are set up, each containing stocks of normal nucleotides plus one dideoxynucleotide
- Each PCR mix should generate all the possible terminating fragments for that particular base
- When the fragments are separated using gel electrophoresis, the base sequence can be determined by ordering fragments according to length
- If a primer is included in each mix, the fragments can be detected by automated sequencing machines
If the Sanger method is conducted on the coding strand (non-template strand), the resulting sequence elucidated will be identical to the template strand
What are the other important functions of DNA except for
coding for proteins?
- Telomeres ( regions of repetitive DNA at the end of a chromosome that protects chromosomal deteriation)
- gene regulation ( sequences that are involved in translation/transcription like promoters)
- Non coding RNA genes ( Codes for RNA molecules that are not translated)
- Tandem repeats of DNA
What are tandem repeats used for?
DNA profilling
Describe the process of DNA profilling using tandem repeats
- Within the non-coding regions of an individual’s genome there are short tandem repeats
- Tandem repeats can be removed using restriction enzymes and then separated with gel electrophoresis for comparison
- As individuals will have different numbers of repeats at a given satellite DNA locus so will generate unique DNA profiles
- Longer repeats will generate larger fragments, while shorter repeats will generate smaller fragments
Nuclesomes
What are the three main components of a gene?
-promoter, coding sequence, terminator
What is the promoter an example of?
Non coding DNA with a function
Name some facts about the promoter
- The non-coding sequence responsible for the initiation of transcription
- Binding site for RNA polymerase (the enzyme responsible for transcription). Binding is controlled by transcription factors.
These transcription factors bind to either near or away from the promoter
What is the anti-sense strand?
- The antisense strand is the strand that is transcribed into RNA
- Its sequence is complementary to the RNA sequence and will be the “DNA version” of the tRNA anticodon sequence
- Can also be referred to as the template strand
What is the sense strand?
- The sense strand is the strand that is not transcribed into RNA
- Its sequence will be the “DNA version” of the RNA sequence (i.e. identical except for T instead of U)
- The sense strand is also referred to as the coding strand (because it is a DNA copy of the RNA sequence)
Describe the process of transcription
- DNA— RNA
- In initiation, RNA polymerase binds to the promoter and causes the unwinding and separating of the DNA strands
- Elongation occurs as the RNA polymerase moves along the coding sequence, synthesising RNA in a 5’ → 3’ direction by linking free nucleotides together
- When RNA polymerase reaches the terminator, both the enzyme and RNA strand detach and the DNA rewinds
What happens to mRNA after transcription in eukaryotic cells?
They are modified
What are the three events after transcription that must occur in order to form mRNA?
Capping
Adding a methyl group to the 5’-end of the transcribed RNA which provides protection against degradation by exonucleases and allows the transcript to be recognised by the cell’s translational machinery (e.g. nuclear export proteins and ribosome)
Polyadenylation
Adding a long chain of adenine nucleotides (a poly-A tail) to the 3’-end of the transcript.The poly-A tail improves the stability of the RNA transcript and facilitates its export from the nucleus
Splicing
Within eukaryotic genes are non-coding sequences called introns, which must be removed prior to forming mature mRNA
The coding regions are called exons and these are fused together when introns are removed to form a continuous sequence
Introns are intruding sequences whereas exons are expressing sequences
The process by which introns are removed is called splicing
What does splicing mRNA do?
Increases the number of different proteins an organism can produce
What type of splicing involves the removal of exons?
Alternative splicing
How does splicing increase the number of proteins?
The selective removal of specific exons will result in the formation of different polypeptides from a single gene sequence
What is gene expression regulated by?
By proteins that bind to specific base sequences
What are the two groups of proteins that regulate gene expression?
Regulatory proteins and transcription factors
What is the function of nuclesomes?
Regulate transcription
What are nuclesomes?
DNA that is wrapped around histone proteins
What is acetylation?
- adding an acetyl group to a histone tail which nuetralises the charge making DNA less tightly coiled ans increasing rate of transcription
What is methylation?
- adding a methyl group to the tail which maintains the positive charge of the tail making DNA more coiled and reducing transcription
What is heterochromatin?
When DNA is supercoiled and not accessible for transcription
What is euchromatin?
When DNA is loosely packed and accessible for transcription
How do tRNA activating enzymes illustrate enzyme substrate specificity?
- Each tRNA molecule binds with a specific amino acid in the cytoplasm
- Each amino acid is recognised by a specific enzyme (the enzyme may recognise multiple tRNA molecules due to degeneracy)
Describe the process of tRNA activation?
- tRNA activating enzyme binds to ATP and a specific amino acid which forms an amino acid complex
2.
Describe the process of tRNA activation?
- tRNA activating enzyme binds to ATP and a specific amino acid which forms an amino acid complex
- tRNA molecule is recruited and binds to the amino acid
- amino acid complex is released
- tRNA is now charged
What is the role of phosphorylation in tRNA activating enzymes?
- The function of the ATP (phosphorylation) is to create a high energy bond that is transferred to the tRNA molecule
- This stored energy will provide the majority of the energy required for peptide bond formation during translation
Describe the structure of ribosomes
- made up of protein and RNA
- small and a large sub unit
- can be free floating or bound to rough eR ( in eukaryotes)
- 70s in prokaryotes and 80s in eukaryotes
Describe the structure of tRNA
- The acceptor stem (3’-CCA) carries an amino acid
- The anticodon associates with the mRNA codon (via complementary base pairing)
- The T arm associates with the ribosome (via the E, P and A binding sites)
- The D arm associates with the tRNA activating enzyme (responsible for adding the amino acid to the acceptor stem)
Describe the process of translation
The first stage of translation involves the assembly of the three components that carry out the process (mRNA, tRNA, ribosome)
The small ribosomal subunit binds to the 5’-end of the mRNA and moves along it until it reaches the start codon (AUG)
Next, the appropriate tRNA molecule bind to the codon via its anticodon (according to complementary base pairing)
Finally, the large ribosomal subunit aligns itself to the tRNA molecule at the P site and forms a complex with the small subunit
Elongation
A second tRNA molecule pairs with the next codon in the ribosomal A site
The amino acid in the P site is covalently attached via a peptide bond (condensation reaction) to the amino acid in the A site
The tRNA in the P site is now deacylated (no amino acid), while the tRNA in the A site carries the peptide chain
Translocation
The ribosome moves along the mRNA strand by one codon position (in a 5’ → 3’ direction)
The deacylated tRNA moves into the E site and is released, while the tRNA carrying the peptide chain moves to the P site
Another tRNA molecules attaches to the next codon in the now unoccupied A site and the process is repeated
Elongation and translocation continue in a repeating cycle until the ribosome reaches a stop codon
These codons do not recruit a tRNA molecule, but instead recruit a release factor that signals for translation to stop
The polypeptide is released and the ribosome disassembles back into its two independent subunits
What is formed during transcription?
A. RNA strand complementary to DNA strand, formed by RNA polymerase
B. DNA strand complementary to DNA strand, formed by DNA polymerase
C. RNA strand complementary to RNA strand, formed by DNA polymerase
D. DNA strand complementary to RNA strand, formed by RNA polymerase
A
