Topic 7 Flashcards
Hershey and Chase
Aim: to find out which molecule was the one of heredity
- bacteriophages grown in radioactive P or S
- DNA contains P, proteins in protein coat contain S
- bacteriophages were allowed to infect bacteria
- mixture was centrifuged
- In the radiolabelled S mixture -> radioactivity was detected in the supernatant
- In the radiolabelled P mixture -> radioactivity detected in the bacteria pellet
Conclude: DNA is the molecule of heredity
How X ray diffraction of DNA conducted
- DNA purified and fibres stretched in thin glass tube -> make most of strands parallel
- targeted with an X ray beam
- X rays hit atoms and get diffracted -> create scattering pattern on film paper
Conclusions:
- DNA is double stranded
- nitrogenous bases packed on the inside, while phosphate backbone on the outside
- double helix structure
The different discoveries leading to the structure of DNA
Franklin:
- dna has a regular structure
- sugar phosphate outer backbone + nitrogenous bases inside
Chargaff:
- equal number of purines and pyrimidines
- the purine and pyrimidines must be paired -> hence antiparallel directions
Watson and Crick:
- the A-T and G-C bond lengths are the same -> 2 H bonds and 3 H bonds respectively for the regular structure
2 mechanisms of replication discovered:
- replication is bi-directional (diff directions as 2 strands run antiparallel)
- CBP occurs
DNA gyrase function
single strand binding proteins function
Gyrase: reduces torsional strain from the unwinding of DNA strands by helicase. Relaxes positive supercoils that would otherwise form
SSB proteins: bind to the strands of DNA to prevent them from re-annealing. Also prevents nucleases from digesting the strands. They are dislodged when the new complementary strand is synthesised
How DNA Poly 3 works
- Cannot initiate a new strand -> will only add free nucleotides to existing strand
- binds to the 3’ end of the template strand
- synthesises new strand in a 5’ to 3’ direction
How it adds nucleotides:
- free nucleotides are present as deoxynucleoside triphosphates
- DNA Poly 3 cleaves 2 phosphates and uses the energy released to form a covalent bond w the sugar phosphate backbone
Explain the Sanger method
Dideoxynucleotides:
- lack the OH group needed to form the phosphodiester bond
- causes the replication to terminate as soon as it is incorporated.
Sanger method:
- DNA is sequenced using dideoxynucleotides
- 4 mixtures set up -> each with all nucleotides and one dideoxynucleotide type.
- PCR –> generates over a billion molecules -> all possible combinations of terminating fragments generated
- the fragments are then separated usign gel electrophoresis -> the sequence of the non-coding (template) strand is generated
Functions of non-coding DNA
Satellite DNA: Used in DNA profiling
Telomeres: at the end of dna molecules -> prevents damage to molecule
Introns: sections of non-coding dna within genes
Non-coding RNA genes: codes for RNA molecules not translated into protein -> eg rRNA tRNA
Gene regulatory sequences: involved in transcription (promoters, enhancers, silencers)
Nucleosome structure (the charge bits)
- octamer of histone proteins + additional histone
- DNA - charge, histone protein surface has + AAs
- histones have N-terminal tails protruding from the structure
- in condensation, N tails link up from adjacent histone octamers -> pull them closer together
Benefits of supercoiling in nucleosomes
- compact structure -> efficient storage
- protects DNA from damage + allows it to be mobile during mitosis
Sections of a gene
promoter region:
- where RNA Polymerase binds
- located just upstream of the desired gene sequence
- NOT transcribed
- Repressor proteins can bind to the promoter region to prevent the binding of RNA Poly
- Transcription factors also mediate and control the binding of RNA Poly to promoters
- transcription factors either bind to proximal or distal control elements to control the transcription of the seq.
coding sequence
- actually contains the gene to be coded
- RNA poly unwinds the DNA strands after binding to the promoter region
- synth the mRNA strand in a 5’ to 3’ direction
terminator region:
- after coding sequence
- termination mech varies between eukaryotes and prokaryotes
Sense v antisense
Antisense:
- template strand
- IS transcribed
Sense:
- coding strand
- is NOT transcribed
Transcription process
- Initiation: the RNA Poly binds to the promoter region, causes the unwinding of DNA strands
- Elongation: RNA poly synthesises the mRNA chain in a 5’ to 3’ direction. Free nucleoside triphosphates are joined by phosphodiester bonds. Sequence determined using the template strand and CBP
- Termination: the rna poly reaches the terminator region and separates from the DNA strands. nascent mRNA separates and DNA rewinds
3 ways mRNA is modified after transcription
- poly-A-tail/polyadenylation: a long series of A bases added to the 3’ end of the mRNA sequence -> gives it stability and facilitates its export from the nucleus
- methyl-capping: methyl group added to the 5’ end, to protect it from being degraded by exonuclease, allows translational machinery to recognise
- splicing: introns removed and exons fused together
alternative splicing: introns and SPECIFIC exons removed -> increase the different types of polypep seq. that can be generated
Gene expression regulation - proteins
- Transcription factors must form a complex with RNA poly at the promoter for transcription to occur -> levels regulate transcription rate
- repressor proteins bind to silencer sites -> prevent complex formation
- activator proteins bind to enhancer sites -> mediate complex formation
They decrease and increase the transcription rate, resp., by binding to sites outside of the promoter region
TF presence can be tissue-specific -> varying gene expression rates for diff tissues w diff functions. the reg. protein levels can be increased/decreased by hormone/chemical signals
Control elements
- where reg proteins bind on the DNA seq.
- TFs tend to bind to proximal control elements
- other reg proteins tend to bind to distal control elements
