protein synthesis - mechanisms Flashcards
what is the role of dna in gene expression
1) encodes all genes in an organism
2) can be replicated as information to rna (ie to form proteins)
what is an important mediator in protein synthesis (translation)
transfer RNA (tRNA) also called aminoacyl-tRNA
what is the structure of tRNA molecules
single stranded
1 amino acid covalently bound to the 3’ end via the CCA (cytosine cytosine adenine) arm
anticodon loop contains the anticodon
what is the property of the amino acid in tRNA molecules and what does this mean
amino acylated
so bound to the nucleotide
what is the anticodon in tRNA
- a base triplet
- template-recognition site (recognises codon on mRNA)
how do tRNAs bind to mRNAs
- via their anticodon binding to codon on mRNA
- brings amino acid to right position in the genetic code for protein synthesis
why is the secondary structure of tRNA distinct
- single stranded so folds back on itself
- base pairs with itself by INTRAmolecular base pairing (bases in the sequence are self-complimentary)
- to form anticodon loop
how is the amino acid in tRNA molecules amino-acylated
- adenine is the last base of the CCA arm on tRNA
- amino acid bound to the 3’ hydroxyl group of the terminal adenine via esterification
- forms aminoacyl tRNA loaded with correct amino acid
we always read a genetic code in…
triplets
what is each amino acid on a base sequence encoded by
groups of 3 nucleobases (nucleotide codes) starting from a fixed point
what are base sequence triplets in dna/rna translated to
correct corresponding amino acid ie ABCDEFGHI ABC = aa1 (amino acid 1) DEF = aa2 GHI = aa3
what 3 properties does the genetic (triplet) code have
redundant
unambiguous
universal
what is meant by redundancy of the genetic code
- only 20 amino acids make up most proteins
- BUT 64 possible triplet codons (variants of 4 bases combined to triplet sequence = 64 possibilities) to code for those
- SO several different triplet codon sequences can encode for the same amino acid
what is meant by unambiguity of the genetic code
- each codon can only encode 1 specific amino acid (or START or STOP)
redundancy compensated for by every tRNA being v specific for its encoded amino acid
what is meant by universality of the genetic code
- genetic code is universal
- all known living organisms use the same genetic code based on the same chemistry (common evolutionary ancestry)
what is scientific procedure is universality of the genetic code a fundamental concept for
genetic engineering
- can take genes from one organisms and transfer it to another
- the second organism still understands the code and is reprogrammed with the transferred gene
in a codon, what end is the
a) first base (1st position)
b) second base (2nd position)
a) 5’
b) 3’
what two codon tables do we have
1) translate mRNA into protein directly, has U instead of T
2) same genetic code as 1 but written for dna, T instead of U
what can be observed if we look at mitochondria
- preferential codon usage
- exceptions in standard codon codes
- code in mitochondria different from universal genetic code
why is the code in mitochondria different from universal genetic code
- derived from prokaryotes that have been taken up by the precursor eukaryotic cells
- so have their own translation machinery and genes
other than mitochondria where else is preferential codon usage visible
for the nuclear genome
what codons are different in mitochondria compared to the standard code
1) UGA = stop code in standard, Trp in mito
2) AUA = Ile standard, Met in mito
3) AGA + AGG = Arg standard, stop in mito
what codons are shown in green in genetic code tables
preferential codon
what codons are shown in red in genetic code tables
low usage codons
what is a good example of species-specific differences in specific tRNA anti codon abundance for same amino acid
between homosapiens and yeast
- same genetic code
- different preferential codon usage (due to redundancy of genetic code)
so why would there be problems if we transfer a gene from human to yeast
- yeast understands code
- BUT takes longer to translate the protein
- compensate for this in synthetic biology by optimisation process
how is translation initiated
1) mrna molecule
2) start codon (AUG/ATG)
3) codes for start amino acid methionine (Met)
4) this is a clear initiation signal
how is the mRNA binded to the ribosome to create the translation initiation sequence
1) purine-rich sequence at -10 position of mRNA seq
2) base pairs with rna molecule in the ribosome
3) rna rna base pairing binds the 2 together
what is the translation initiation sequence called
shine dalgarno sequence
what is a clear signal to the ribosome that protein translation needs to start
combination of the -10 initiation seq and +1 start codon
how do reading frames work
- specified by start codon and strength of ribosome binding site
- 3 reading frames are possible in a 4 letter encoded triplet decoded sequence (can start reading triplets from very 1st codon, 2nd codon or 3rd codon)
- giving very distinct different amino acid sequences
what can a mutation lead to
- reading frame shift
- change protein codon sequence even though only 1 base changed
- get completely different protein (ie in insertions and deletions not divisible by 3)
what does protein synthesis require
translation of nucleotide sequences into amino acid sequences
which enzymes load the correct tRNAs with the correct corresponding amino acid thus govern correct translation of the genetic code
aminoacyl-tRNA synthetases
which catalytic particles in the cell are responsible for translating tRNA into proteins by the use of tRNAs loaded w amino acids
ribosomes
what are prokaryotic ribosomes like
70s
- 1 small 30s sub-unit
- 1 large 50S sub-unit
- sub-units are the secondary structures of the protein components of the ribosomal subunit
- made up of large amount of structural ribosomal rna
what does the s correlate to in ribosome structure (ie 70s)
- sedimentation coefficient in centrifugation
- unit of sedimentation speed of ribosomal subunits
- why 30s and 50s = 70s in combination (+ not 80)
what are ribosomes
hybrids of rna and protein called ribonucleoprotein particles
what is contained in the site of peptide bond formation in ribosomes
- only RNA
- no protein
- within 20Å
proteins are synthesised by…
successive addition of amino acids (carried by tRNAs) to carboxyl terminus of previous amino acid
+ subsequent peptide bond formation
what is the rate of and error rate of translation in e. coli what does this mean for protein synthesis
- 40 amino acids/sec
- 10^-4 = error in protein synthesis every 10,000 amino acids
- good fidelity, high probability of synthesising correct protein, even for very long proteins
what would it mean for protein synthesis if the error rate was 10^-2
- 1 wrong amino acid every 100 amino acids incorporated (only get 36% of proteins without error)
- okay for short proteins
- not possible to synthesise 1000 amino acids without making a mistake
what are the most important regions of a cloverleaf tRNA molecule
1) amino acid attachment site (cca arm) at top
2) anticodon loop containing anticodon at bottom
3) other loops containing strange/atypical nucleobases for structural recognition of the tRNA (ie DHU loop and TvC loop)
how do tRNAs confer high fidelity
- by its structural and base pairing features
- INTRAmolecular base pairing
- number of conserved features of base sequence
what modified nucleosides / nucleobases are contained in tRNAs
1) methylinosine(mI)
2) dihydrouridine(UH2)
3) ribothymidine (T)
4) pseudouridine (ψ)
5) methylguanosine (mG)
6) dimethylguanosine (m2G)
7) inosine
what are modified nucleosides / nucleobases important for
- flexibility of codon binding possibilities for the anticodon
what is the significance of INOSINE as a modified nucleoside
- part of anticodon arm
- can base pair with many different bases (C,A,U) compared to standard nucleobases
- important for redundancy and wobble hypothesis
what shape is tRNA likened to if looked at in a 3D way
L shape
- CCA stem at one end of arm
- anticodon at other
how is the 3D L shape of tRNA formed
- INTRAmolecular base pairing forms 3d structure, locks it in place and gives stability
why are aminoacyl tRNA synthetases not called synthases
they require energy for this process of loading corresponding amino acids
what do aminoacyl tRNA synthetases have to do
enzymes that attach amino acids to tRNAs
highly specific for each amino acid
read anticodon of tRNA they have to load and differentiate between amino acids with similar structure
how can aminoacyl tRNA synthetases correct wrongly charged/attached tRNAs
- proofreading function
- they hydrolyse and remove them
how do aminoacyl tRNA synthetases attach amino acids to tRNA
aminoacylation
explain the editing function of aminoacyl-tRNA synthetases (how it recognises the correct amino acid)
- the synthetase enzyme has 2 sites; editing site and activation site
- flexible CCA arm moves amino acid between the 2 sites
- if amino acid fits into editing site = removed via hydrolysis
- if amino acid only fits into activation site = attached to tRNA
what is rRNA (ribosomal)
major structural component of each ribosomal subunit
what is the structure of 16s rRNAs
- extensive secondary structure
- intramolecular base pairing leads to stem loop structures in the same molecule
- x-ray crystallography determines 3d structure
what 2 types of mRNA exist
1) polycistronic
2) monocistronic
what is possible in ribosomes on prokaryotic mRNA but not eukaryotic
ribosome on prokaryotic dna initiates translation of a protein then reinitiates downstream again for another round of protein synthesis of a different protein
all proteins it synthesises are encoded on the same mRNA
why cant ribosomes reinitiate translation in eukaryotic mRNA
every mRNA only encodes 1 protein
so each protein in genome coded for by a distinct mRNA
how is the ribosome able to reinitiate translation in prokaryotes
- prokaryotic mRNA contains multiple different reading frames for different proteins
- structure = binding site -> start codon -> reading frame making protein alpha -> another binding site + start codon for protein beta etc
what are polysomes
- 2+ ribosomes translating mRNA sequence simultaneously
- enable transcription and translation to occur simultaneously
- mRNA nascent to dna its encoded by is synthesised and translation occurs at same time
- specific to prokaryotes bc they dont have nuclei
what is the shine dalgarno sequence
- rna rna hybridisation between mRNA start seq and rRNA
- upstream of the start codon
what site is present on mRNA in addition to the translation initiation site
- site responsible for binding to ribosomes via interaction with 16S rRNA (UAAGGAGGU at -10 upstream of start codon)
- mediate base pairing between rna of ribsome and mRNA
- stabilises structure
- allows initiation of translation
where do prokaryotes show similarity in gene sequence
region upstream of start codon that pairs with the rna sequence in the ribosome
what mediates initial assembly of the translation complex on mRNA
initiation factors
what mediates the process of attaching an incoming amino acid to the growing polypeptide chain and proof reading
elongation factors (ensure high fidelity)
which elongation factor is very important for translation
EF-Tu
- binds to a tRNA
- delivers aminoacyl-tRNA to the ribosome
what 3 tRNA binding sites exist in prokaryotic dna and where
1) E site (exit site)
2) P site (site of peptide bond formation in ribosome)
3) A site (acceptor site = new tRNAs enter as ribosome moves along seq)
- they bridge across the 30s and 50s subunits
how is the growing polypeptide chain (protein) extruded from the ribsome during translation and released from when translation is complete
through a tunnel which passes through the 50S subunit
what is the action of initiation factor 1 in translation initiation (prokaryotes)
mediate assembly of 30S sub unit
what is the action of initiation factor 2 in translation initiation (prokaryotes)
forms 30S initiation complex
by bringing the 1st tRNA (that recognises start codon + is amino acylated w methionine)
what do initiation factors do following the formation of the 30s complex
- 50s subunit assembled to 30s
- forms 70s initiation complex
- translation can start
so translation initiation is assembly of fully functional ribosome made up of a small and large subunit on the mRNA which has to be translated
how does translation occur on a ribosome
1) peptidyl-tRNA in P site
2) aminoacyl-tRNA binds in A site
3) new peptide bond formed as both sites occupied
4) translocation frees up A site
5) deacylated tRNA dissociates
what does the elongation factor EF-G do
catalyses recycling of ribosome
ie once it has formed a peptide bond the ribosome moves along the mRNA to move the old tRNA into its P site and free up its A site the EF-G alters it shape so its ready to accept the next charged tRNA
what do enzymes have for for energy and conformational change
GTP molecules
- phosphorylate is to release energy (GDP + Pi)
- both elongation factors have these
what does EF-TU do
1) check for correct base pairing between codon + anticodon
2) brings new tRNA to catalyse formation of peptide bond
what is the action of EF-Tu if base pairing is
a) incorrect
b) correct
a) rerelease of tRNA elongation factor compound from the ribosome
b) switch in EF-Tu turned on
- GTP hydrolysed to GDP and released releasing the tRNA amino acid complex for the action of the ribosome
what is the wobble hypothesis
- dictates allowed pairings at the 3rd base of the codon
- tRNA has flexibility in 3rd base of codon (at 3’ end) to bind multiple codons and deliver the same amino acid
- responsible for redundancy
why does tRNA have this flexibility in its 3rd codon base
atypical nucleobases on 1st position of anticodon arm (‘wobble position’) have flexibility in binding (can bind multiple different nucleotides)
in bacteria what are the possible anticodon bases for the following wobble codon bases U C A G
U = A,G, I C = G, I A = U, I G = C, U
in eukaryotes what are the possible anticodon bases for the following wobble codon bases U C A G
U = A,G, I C = G, I A = U G = C
if the 1st base of the anticodon was
C, A, U, G, I
what would the 3rd base of the codon be
C = G A = U U = A, G G = U, C I = U, C, A
what is enabled if the first base of the anticodon is I
- almost full flexibility of tRNA to bind (U C or A)
explain why leucine is more complex in regards to its wobble flexibility
- has 1 tRNA that can bind CUA, CUC, CUG, CUU codons because they only differ in the wobble base
- but also has 2 additional codons (UUA, UUG) very different in initial bases
- so a DIFFERENT trna also delivers leucine but recognises a different codon
which antibiotics inhibit protein synthesis
1) streptomycin + other aminoglycosides
2) tetraccycline
3) chloramphenicol
4) cycloheximidine
5) erythromycin
6) puromycin
how can antibiotics mediate killing in bacteria (prokaryotes only) by inhibiting protein synthesis
1) bc of 3d structural features = get into cell, into ribosome, bind ribosome in irreversible way that directly interferes with its function shutting its translation down
2) bind small or large ribosomal sub-unit depending on their chemical composition and structure
what happens when antibiotics shut down protein translation
- no longer synthesise proteins essential for survival
- cell dies
how do streptomycin + other aminoglycosides inhibit protein synthesis
- inhibit initiation
- cause misreading of mRNA
- pro only
how does tetracycline inhibit protein synthesis
- binds to 30s subunit
- inhibits aminoacyl tRNA binding
- pro only
how does choramphenicol inhibit protein synthesis
- inhibit peptidyl transferase activity of 50s
- pro only
how does erythromycin inhibit protein synthesis
- binds to 50s
- inhibits translocation
- pro only
how does cycloheximidide inhibit protein synthesis
- inhibit peptidyl transferase activity of 60s
- euk only
how does puromycin inhibit protein synthesis
- cause premature chain termination by acting as analogue of aminoacyl tRNA
- pro + euk
