Week 6 Flashcards
3’ end processing
- consensus sequences direct cleavage and polyadenylation of the 3’ end
- 3’ end processing proteins move from CTD to mRNA
- cleavage and addition of a poly-A 3’ tail along with Poly A-binding proteins result in the mature mRNA
state 4 features of the genetic code
- universal for almost all genomes
- codons are read as mRNA triplets encoding all 20 amino acids
- redundancy: multiple codons for most amino acids
state the stop and start codons
AUG - methionine - start
UAA/UAG/UGA - stop codons (don’t code for an AA)
describe the possible mutations that could occur within a reading frame
- nucleotide-pair substitution: silent (the amino acid does not change)
- nucleotide-pair substitution: missense (the amino acid is changed into a different amino acid)
- nucleotide-pair substitution: nonsense (leads to a premature stop codon)
- 1 nucleotide-pair deletion (frameshift causing immediate missense - all following amino acids likely to be wrong)
- 1 nucleotide-pair insertion (frameshift causing immediate nonsense)
- 3 nucleotide-pair deletion (no frameshift, but amino acid changes)
why is the cloverleaf appearance of tRNA so important?
- tRNA acts as an adaptor molecule
- its secondary structure is critical to RNA function
- the anticodon loops allows the anticodon to base pair with the mRNA codon in a complementary and antiparallel way
draw a standard tRNA structure and label it
are there the same number of tRNAs as there are anti-codons?
if we take out the stop codons, we have 61 possible anti-codons, but bacteria have 31 tRNAs and humans have 48.
two possible strategies:
- more than 1 tRNA for many amino acids
- some tRNAs can recognise and base pair with more than one codon
describe the wobble position of a tRNA molecule
- third base pair between codons/anticodons
- flexible: base pair does not have to be perfect
- saves the number of tRNAs that have to be produced
- may ensure mutations have a lesser effect
wobble codon base and possible anticodon bases in bacteria
U = A, G, I
C = G, I
A = U, I
G = C, U
wobble codon base and possible anticodon bases in eukaryotes
U = A, G, I
C = G, I
A = U
G = C
what is I?
Inosine, which represents a post-transcriptional modification of adenosine
seen the presence of a wobble position, how fidelity in base pairing between codons and anticodons ensured?
- sequential steps in ensuring fidelity:
- aminoacyl-tRNA synthetases: check compatibility of amino acid and tRNA then makes a high-energy bond using ATP
- base pairing between mRNA and tRNA in ribosome - error correction by aminoacyl tRNA synthetase:
- by hydrolytic editing to break the high energy between the tRNA and amino acid
how is recognition of a specific tRNA by its synthetase achieved?
- identifying the tRNA anticodon nucleotides
- recognising the nucleotide sequence of the acceptor stem/arm
- reading nucleotide sequences at additional positions on the tRNA
label a diagram of tRNA synthetase binding to tRNA
location of ribosomes in a eukaryotic cell
- on endoplasmic reticulum
- in cytosol
location of ribosomes in a prokaryotic cell
- in cytosol
distinguish between the function of the large and small ribosomal subunit
The small subunit (40S in eukaryotes) decodes the genetic message and the large subunit (60S in eukaryotes) catalyzes peptide bond formation.
how is peptide synthesis made energetically favourable?
with the energy stored in covalent bond between the amino acid and the tRNA in P site
A, P, E site
A site - aminoacyl site - aminoacylated tRNA enters here
P site - peptide site - where the peptide bond is formed
E site - exit site
what catalyses the formation of the peptide bond?
peptidyl transferase activity of the rRNA in the large subunit - ensures the high energy bond between the amino acid and the tRNA provides the energy for forming the new peptide bond
which translocates first - the large or the small ribosomal subunit?
the large subunits translocates first; then, the small subunit
describe the structure of a prokaryotic ribosome (eukaryotic is very similar)
- ribosome is a ribozyme
- L1 protein is involved in folding and stabilising RNAs
- 5S RNA: component of the large ribosomal subunit thought to enhance protein synthesis by stabilization of a ribosome structure
- 23S rRNA: for tRNA binding in the P site of the large ribosomal subunit
elongation factors
- EF-Tu (pro) / EF1 (eu) checks aminoacyl tRNA.
- if base pairing is not correct, EF-Tu is not released and peptide bond can’t form.
- if base-pairing is correct, GTP is hydrolysed and EF-Tu is released
- there is also a slight delay before the formation of the peptide bond which allows one last check for accurate base pairing - EF-G (pro / EF2 (eu) helps the ribosome to move the mRNA forward one codon and helps speed up elongation of the polypeptide chain
- requires energy from hydrolysis of GTP-> GDP
- without this EF, translation is very slow
can ribosomes perform protein synthesis without the aid of elongation factors?
yes, but it is much slower, more inefficient, and less accurate
distinguish between mRNA structure in bacteria (prokaryotes) and eukaryotes
- Prokaryotic mRNA has a Shine-Dalgarno sequence for ribosome binding, while eukaryotic mRNA has a 5’ cap and poly-A tail for protection and ribosome binding.
- The first amino acid differs: formylmethionine in prokaryotes and methionine in eukaryotes.
- mRNA is polycistronic in prokaryotes (produces multiple proteins) and monocistronic in eukaryotes
initiation of translation in prokaryotes
- shine-dalgarno sequences on mRNA base pair with rRRNA in small ribosomal subunits
- positioning of small ribosomal subunits to initiating AUG codons on mRNA also requires Initiation Factors (IFs)
- fMethionine aminoacyl tRNA binds to initiator codon in the P-site of small ribosomal subunit. Formyl group has been covalently added to R group of Met.
- large ribosomal subunit binds
initiation of translation in eukaryotes
- The initiator tRNA, charged with methionine, and various translation initiation factors, bind tightly to the P site of the small ribosomal subunit.
- The small ribosomal subunit attaches to the 5’ end of an mRNA molecule and scans along the mRNA until it encounters a start codon (AUG).
- Upon finding this start codon, translation initiation factors dissociate and the large ribosomal subunit binds
- a charged tRNA binds to the second codon
- first peptide bond forms
function of initiation factors that bind at the 5’ cap and poly-A tail of the mRNA in eukaryotes
- these can bind to each other, bringing the 5’ end closer to the 3’ end, thus circularising RNA
- checks both ends to ensure modification has been correct and RNA is ready for translation
- facilitates ribosome recycling and enhances translation efficiency by making it easier for ribosomes to reinitiate translation on the same mRNA molecule
how is translation terminated?
- there is binding of a release factor to the A site, which breaks the bond between the tRNA and amino acid in the P site
- hydrolysis causes the polypeptide chain to be released and the ribosome dissociates
is the human translation release factor a protein or a tRNA?
eRF1 (in eukaryotes), RF1, and RF2 (in prokaryotes) are proteins
polyribosomes/polysomes
- arises from circularising RNA
- makes translation much more efficient
- occurs in both eukaryotes and prokaryotes
- multiple ribosomes attached to the same mRNA strand to produce multiple proteins from the same mRNA
- otherwise, protein synthesis is relatively slow
- ribosomes are spaced every 80nt
how is protein folding conducted?
- most proteins can fold on their own but this is usually incorrect and slow
- chaperone proteins - Hsp60 and Hsp70 - are used to help correctly fold the proteins
post-translational modifications
- many proteins require post-translational modifications such as phosphorylation and glycosylation (common in membrane proteins)
- covalent modifications may be required to make a protein active or recruit a protein to the correct membrane/organelle
when do proteins need to be degraded?
- haven’t folded properly
- amino acids have been modified
describe protein degredation
proteins targeted for degradation have a small protein called ubiquitin covalently attached to them, which directs them to the proteasome where they are degraded by proteases, and the amino acids recycled into new proteins by the cell
describe the structure of a proteasome
- polyubiquitin-binding site
- central cylinder (containing active sites of proteases)
- stopper
composition of a ribosome
two thirds RNA and one third protein by weight
main role of the ribosomal proteins
- to help fold and stabilise the RNA core, while permitting the changes in rRNA conformation the are necessary for this RNA to catalyse efficient protein synthesis
how is the catalytic site for peptide bond formation on ribosomes formed?
- by the 23S rRNA of the large subunit
- peptide transferase
- highly structured pocket that precisely orients the two reactants - polypeptide and incoming amino acid - increasing the likelihood of a productive reaction
how do most antibodies work?
act bye inhibiting bacterial, but not eukaryotic, gene expression.
