translation Flashcards
translation =
mRNA-dependent protein synthesis where mRNA is used as a template and the enzyme is the ribosome
translation occurs in the…
cytoplasm of eukaryotes
coupled with transcription in prokaryotes
eukaryotic mRNA generated by transcription -
has a single open reading frame (coding sequence) with untranslated regions on either end (5’ and 3’)
eukaryotic translation starts at the…
start of the open reading frame, however, the ribosome doesn’t bind directly to the start site, it binds to the 5’ cap
the ribosome moves…
from the cap and scans the untranslated region to find the start codon at the beginning of the open reading frame
the ribosome doesn’t make anything whilst it scans - it starts reading from the start codon (methionine AUG)
bacteria mRNA =
polycistronic = has multiple translation start sites
protein synthesis occurs on…
polyribosomes
ribosomes bind in sequence from…
5’ start end to the 3’ end
the ribosome goes the whole length of the mRNA, taking its nascent peptide with it an elongating that peptide chain as it goes along
start codon =
AUG
methionine = always the first amino acid at the N-terminus of the growing peptide chain
in some bacterias, GUG is the start codon = valine amino acid which can be used to insert methionine
the peptide chain grows at the…
carboxy-terminus on the ribosome, as the ribosome moves from 5’ end towards 3’ end
stop codon =
UAG
UGA
UAA
transfer RNA:
takes specific amino acid to the ribosome for protein synthesis
at least one tRNA is dedicated to one specific amino acid, although some amino acids have several cognate tRNAs
tRNA contains ‘odd’ nucleosides:
when ribosome gets added to N1 - you get the normal nucleoside uridine
- in all tRNAs: in some cases, Pseudo uridine forms = abnormal rotated base
…due to pseudoisomerase enzyme rotating the base creating an isomer in which uracil is attached to the ribosome via a carbon-carbon at C5 instead of a nitrogen-carbon glycosidic bond at N1
another form of modification of the uracil base is a reduction - in which Dihydrouracil is formed = not a flat ring
secondary structure of tRNA:
- folds into a common secondary structure comprising ~76 nucleotides
- is single-stranded and folds back on itself to form stem-loops with base pairing in the helical stem
- 5’ end carries a monophosphate
- there are 4 helical stem-loops:
DHU-loop = consists of residues of dihydrouracil
anticodon stem-loop = at nucleotides 34, 35, 36
variable stem-loop
T-lop = consists of pdeudo uridine - at nucleotides 54, 55, 56 - 3’ amino acid attachment site (CCA terminus) = at nucleotides 74, 75, 76
tertiary structure of tRNA:
get from secondary structure to the tertiary structure via a process called co-axial stacking to get a characteristic L shape of the tRNA molecule
- unpaired nucleotides in tRNA secondary structure brought together in folded tertiary structure
- tertiary pairs form between partners in the T-loop, D loop and variable stem-loop
- T-loop and D-loop are close in tertiary structure = interactions between unpaired nucleotides in T-loop and D-loop = co-axial stacking
codon-anticodon interctaion:
when tRNA goes into the ribosome it encounters mRNA and undergoes codon-anticodon recognition:
- 3 bases in mRNA codon pair to 3 bases in tRNA anticodon (position 34, 35, 36)
- Watson Crick pairing at first two positions = mRNA codon position 1 and 2 / tRNA anticodon position 2 (35) and 3 (36)
- alternative pairing at mRNA codon position 3 - tRNA anticodon position 1 (34) = wobble pairing
wobble pairing =
- more relaxed form of base pairing where G-U pairs are likely
- G-U mismatch requires wobble geometry
- tRNAs can recognise multiple cognate codons if they have G, U or I in the appropriate position
aminoacyl-tRNA synthase:
aaRS = enzymes that attach appropriate amino acids onto cognate transfer RNA molecules
there are one of these enzymes per amino acid (20 types)
they are named after the cognate amino acid - using the 3 letter abbreviation (e.g. LysRS)
two step process of aminoacyl-tRNA synthases:
1) activation of amino acid:
- the amino acid binds to ATP and the synthase to form an aminoacyl-adenylate, releasing inorganic pyrophosphate
2) charging of cognate tRNA:
- the amino acid from the aminoacyl-adenylate binds to the appropriate tRNA molecule and the synthase gets recycled
how aminoacyl-tRNA synthases find the correct amino acid:
- the amino acid gets added onto the ribose of the terminal adenosine residue (on the CCA terminus) at the end of the tRNA molecule
- aaRS recognises R group of amino acid = have a specific active site to activate a certain amino acid
if an aminoacyl-tRNA synthase is presented with an incorrect amino acid:
false activation of non-cognate amino acid
the tRNA that is cognate for the correct amino acid picks up (with 100% efficiency) the false activated amino acid = forms a false charged intermediate
the false charged intermediate can either:
- false product dissociates from the synthase (non-cognate product forms)
- hydrolytic editing = false intermediate is recognised and cleaved
Fersht ‘double sieve’ model:
the only way to get efficient activation of cognate amino acids is if there was a two-step process = meaning aaRS enzyme must have two active sites:
- activation site
- editing site
the flexible CCA arm of an aminoacyl-tRNA can move the amino acid between the activation site and the editing site
the amino acid is added onto the end of the tRNA molecule in the activation site - if the amino acid fits well into the editing site, the amino acid is removed by hydrolysis
if the incorrect amino acid is isosteric (same size) to the cognate amino acid, then this normally isn’t a problem as they will be chemically different due to polarity
elongation factor:
carrier protein which takes aminoacyl-tRNA to a ribosome for binding
e. g. elongation factor Tu (GTP binding protein in bacteria):
- when it binds to the ribosome GTP gets hydrolysed and the protein comes out with GDP bound to it
- Tu binds GDP more tightly so a helper protein (Ts) is required to displace the GDP and then gets displaced itself by GTP
the aa-tRNA, elongation factor and GTP complex:
binds to the A site of the ribosome mRNA complex (via codon-anticodon recognition)