Transcription and translation Flashcards
DNA requirements to be genetic material
-Must carry information
-Must replicate
-must allow info to change-mutation
-must govern the expression of the phenotype-gene function
Stores information in sequence of bases
What does LDL cause?
If people have this single base pair change, change the sequence of amino acids asn to serine
What is serine associated with?
- High cholesterol
- high LDLS
- coronary heart disease
What is an example of personalised medicine?
CYP 2D6 gene sequence
- amino acid changed to stop codon, enzyme cannot break down certain drugs
- doctors then can prescribe a different tailored drug for patient
In replication what keeps DNA from annealing?
-Single-Stranded DNA proteins
Who discovered the mechanism in biological synthesis of DNA replication?
Kornberg in 1959
He discovered the process of complimentary base pairing and how it could happen
What is the mechanism of DNA replication?
- Initiation-proteins bind to DNA and open up helix, prepare DNA for complimentary base pairing.
- Elongation-proteins via DNA polymerase connect sequences of nucleotides into continuous new strand of DNA.
- Termination-proteins released the replication complex.
What are the rules of DNA Replication?
-Semiconservative – starts at DNA origin – can be uni or bidirectional -semi discontinuous – always 5’-3’ direction -RNA primers required
What is semiconservative replication and who discovered it?
Meselson -Stahl Proved half of DNA comes from each parent
Where does DNA replication start from?
Starts at an ORI sequence
- Initiator proteins identify specific base sequences of DNA called sites of origin
– highly conserved
-prokaryotes have single ori sites eukaryotes have multiple ori sites
What is the difference between replication in bacteria and humans?
- In bacteria much faster application
- enzymes not as good as proofreading
- replication in humans quicker if multiple firing of ori sites
- more accurate in humans
- initiation proteins bind to initiator sites
What is semi discontinuous replication?
- Antiparallel strands replicated simultaneously
- leading strand synthesis continuously in 5‘-3‘ direction
- lagging strand synthesis in fragments in 5’-3’ direction
- Lagging strand is slower, moving in opposite direction to fork, synthesis done in OkaZaki fragments
What is an Okazaki fragment?
-Short sequences of DNA nucleotides which are synthesised discontinuously later linked by DNA ligase to create lagging strand
Why does DNA replication only occur in five prime to 3 prime direction?
- Breakdown of phosphate provides energy
- must break triphosphate down for replication to work - only can occur in 5’-3’ direction
- If it was 3’-5’ enzyme would get added but subsequent dNTPs is cannot bind, they are blocked
What is base mismatch?
- Polymerase has multiple subunits involved in proofreading that right bases are added
- it will then remove mismatched bases
- this is why replication is slower in Eukaryotes
Why are RNA Primers required for DNA synthesis?
- RNA primer required, lay down for DNA replication to occur
- done by a primase
- One primer on leading strand, loads of primer is on lagging as every time DNA is added needs a primer
Function of topoisomerases
Prevents torsion by DNA breaks
Function of helicase
Separates two strands
Function of primase
RNA primer synthesis
Function of single-stranded binding protein
Prevents re-annealing of single strands
Function of DNA polymerase
Synthesis of new strand
Function of tethering protein
Stabilises DNA polymerase
Function of DNA ligase
Phosphodiester bond linkage
In prokaryotes the genes are usually…
Switched on – codes for components for production of tryptophan
-gene is transcribed in operons
In eukaryotes genes are usually…
- Switched off
- not all genes code for proteins
Where does transcription occur in prokaryotes? And what occurs? Eg. Tryptophan
Cytoplasm due to lack of nucleus
Tryptophan -
- Transcription starts at one point and transcript is made from operon
- Entire transcript made in one go in operon
- genes are switched on
Features of transcription in eukaryotes
- Linear-each gene responsible for tryptophan on different chromosomes
- gene is usually switched off
- series of mRNA transcripts which are then translated into proteins
- making of mRNA transcript occurs in nucleus –Primary transcript
- mRNA-out of nucleus for translation
- not all genes code for protein
What does micro RNA do?
Bonds to another RNA molecule and destroys it so that no protein is made
Where does the untranslated regions lie?
In between initiation codon
- its not translated
What are the regulatory regions in transcription?
- Tata box
- GC box
- Order in which boxes placed tell you this is regulatory region
- highly conserved regions dictate whether transcription should occur
What are the coding regions of DNA in transcription?
- AG and GT at beginning and end of exon, the bits inbetween are introns
- All species have methionine at start (ATG)
- At end highly conserved stop codons
- base sequence at end is AATAA
What are the three stages in the mechanism of transcription?
Initiation
Polymerisation
Termination
What occurs in initiation in transcription?
RNA polymerase binds to promoter Along with transcription factors and opens helix
What occurs in polymerisation in transcription?
De novo synthesis using rNTPs as substrate
- chain elongation in 5’-3’ direction
What occurs in termination in transcription?
Stops at termination signal, stop codons
-one stand always a template
What occurs in maturation of primary mRNA transcript?
- RNA capping
- polyadenylation
- splicing
-end up with mRNA transcription with cap, polyadenine tail and no introns
What is capping?
- Addition of 7-methyl-guanosine at 5’ end
- Mediated by guanylyltransferase
What does capping do?
-mRNA highly prone to degradation, mRNA highly unstable compared to DNA
-In protects against the degradation, serves as recognition site for ribosomes, transport mRNA out.
– MRNA goes via nuclear pore into cytoplasm using enzymes
What is tailing?
- Addition of Poly(A) residues at 3’ end
- Driven from energy from ATP
How is the poly a tail attached?
- Poly(A) polymerase, cleavage and polyadenylation specificity factor attaches Poly(A) generated from ATP
- Add series of A’s to 3’ end
What is splicing?
-Removal of introns by snRNPs and spliceosomes
How does splicing occur?
- One cut made, loop made from free-floating end, second cut from route of Lariat to splice intron out
- recognise Exon intro boundaries
- Once spliced you have mature RNA
What does alternative splicing allow?
Multiple transcripts from a single gene
What are housekeeping genes?
-Genes which are made all the time e.g. tubulin
What three things does translation require?
1) mRNA
2) aminoacyl-transfer RNA (aatRNA)
3) Ribosomes
In translation what do ribosomes form?
A catalytic domain into which tRNAs enter with the attached amino acids
What does degenerate mean?
More than one triplet per amino acid
Features of tRNA molecule
- One tRNA per amino acid
- tRNA must be charged
- D loop, T loop, has amino acid tagged onto three prime end
- Two active sites, one for the amino acid one for tRNA
What must be done to tRNA?
1) Covalently attached correct amino acid to tRNA specified by anti codon
2) select the correct charged tRNA specified by mRNA
- coupling of correct amino acid done via aminoacyl tRNA synthetases
When selecting the correct amino acid (done via aminoacyl tRNA Synthetases) what doesn’t the 3rd base have to do?
Conform to Watson Crick base pairing
What occurs in initiation in translation?
STAGE 1 - FORMATION OF 43S pre-initiation complex
-mRNA sequence signals where protein synthesis begins
- 40s-eIF3 bound by eIFA to ternary complex of tRNA (methionine, eIF2 and GTP)
-First amino acid always methionine, energy from GTP not ATP
– Cofactor 2 bound to GTP
-Anti codon loop recognises its complement
What occurs in initiation in translation?
STAGE 2 - FORMATION OF initiation complex
- cap Binding of mRNA to 43S
- mRNA has cap and tail, have protein is bound to them called PABP, bind on 3’ end of sequence to A’s
- Initiation factor complex comes in (eIF4) and binds to 5’ end of cap
- Within complex head and tail link to form a loop
- allows for charged methionine to bind
What occurs in initiation in translation?
STAGE 3 - Positioning at start codon
- Initiation complex unwinds mRNA using eIF4 helicase
- Initiation complex stops at start site AUG
- Recognition allows an irreversible GTP hydrolysis of eIF2 preventing further unwinding
- All secondary structures removed, Helicase to prime apart
What is the highly conserved sequence to allow the complex to know the true AUG start codon?
ACCAUGG KOZAK SEQ
What occurs in chain elongation in translation?
STAGE 1 - aatRNA binds
- aatRNA binds to A site on ribosome by base pairing with codon
- Methionine start codon
- EF1 driven by GTP energy currency, bring correct charged tRNA ahead of the methionine
- 1st initiation methionine called met i
What does EF stand for?
Elongation factor
What occurs in chain elongation in translation?
STAGE 2 - Conformation change
- Confirmation change in the ribosome, induced by GTP hydrolysis of eF1a
- In order for amino acids to form peptide bond with the next amino acid’s ribosome changes shape
- EF1 Dissociates as its job is done
What occurs in chain elongation in translation?
STAGE 3 - Transpeptidation
- C Terminal of polypeptide uncoupled from P site tRNA and peptide bond transferred to amino acid on A site tRNA Catalysed by Peptidyltransferase
- No charged tRNA here
What occurs in chain elongation in translation?
STAGE 4 - Translocation
- GTP Hydrolysis of EF2 to cause second confirmational change
- P site tRNA transferred to E site
- Simultaneous transfer of A site tRNA move to P site
- shape changeX EF2 driven by GTP
What occurs in step three of translation - termination?
- Release factors recognise and bind to stop codons
- induces peptidyl transferase to transfer peptidyl group to water instead of aatRNA
- Uncharged tRNA released from ribosomes
- Inactive ribosome then release mRNA
Where are proteins in translation made?
Polyribosomes
Why does mRNA have a loop confirmation in translation?
Translation increased efficiency
Post-translational modifications-protein folding
- Nascent protein folded
- Mediated by HSP 70/HSP 60 complexes
Post-translational modifications – covalent modifications
-Addition of side groups
-Kinases add phosphate group to certain target proteins
– sidechains added on
– dictate activity/lifespan of proteins
Post-translational modifications - proteolytic cleavage
-Activate some in active precursors
In proteolytic cleavage what do caspases do?
Trigger apoptosis
In proteolytic cleavage what do zymogens do?
Inactive precursors, only activated when cleaved
What is the a site in a ribosome?
acceptor site – binds to aminoacyl tRNA which holds new amino acid to be added
What is the P site in a ribosome?
Peptidyl site-binds to tRNA holding growing polypeptide of amino acid
What is the e site in ribosome?
Exit site-threshold, final transitory step before tRNA now bereft of amino acid is let go by ribosome
Antibiotics that inhibit translation only found in prokaryotes
Streptomycin-prevents initiation elongation
Chloramphenicol-Blocks peptidyl transferase in mitochondria
Antibiotics that inhibit translation only found in eukaryotes
Cycloheximide-Blocks translocation only in cytosol
Antibiotics that inhibit translation found in both prokaryotes and eukaryotes
Puromycin – causes premature release of polypeptide
Which of the following scientists first discovered that the percentage of GC is constant in any given species.
Erwin Chargaff
Small nucleolar RNAs are involved in
rRNA modification and processing
The ability of DNA to denature is important for
DNA synthesis
Nucleic acid hybridization experiments
RNA synthesis
Polymerase Chain Reaction
What occurs in initiation in translation?
STAGE 4 - Association of large subunit (60S)
Irreversible GTP hydrolysis mediates the association of 60S-eIF6 (large subunit ) to the small subunit by the action of eIF5
This becomes the P site
