Translation & Proteins I Flashcards
Translation and proteins
Translation is the biological polymerisation of amino acids into polypeptide chains
- The code is converted to another ‘language’
–> Nucleotide sequences on mRNA to the amino acid
sequences of the proteins
Process requires:
- amino acids
- messenger RNA (mRNA) - carrying triplet code - directs the
order in which the amino acids should be polymerised
- ribosomes –> means of directing amino acids into correct
position…
tRNAs as adapters
Adapter molecule that can covalently bond to the amino acid (on the one end), while also being able to form Hydrogen bonds between the nucleic acids ( on the other end)
= tRNAs
- adapt genetic information present as specific triplet codons in mRNA to their corresponding amino acid
- Anticodons on tRNA bind to complementary codons of mRNAs, bringing the amino acid which is covalently bonded to the tRNA closer and holding it in close proximity to the adjacent amino acid in the sequence = allowing formation of a peptide bond between them
- This process occurs over and over on the ribosome as the mRNA runs through it = allows polymerization of amino acids into a polypeptide
Ribosomes
- Abundant in cell: 10 000 copies in bacterial cells - euk. have
even more - 20 -30 nm diameter
- Large & small subunits
- A single ribosome = (monosome)
Contains:
> rRNA (catalytic function in translation = actually ribozymes)
> ribosomal proteins (fine-tuning of translation) - High degree of Redundancy in rRNA genes
- Polycistronic transcription of rRNA
> found in both Prokaryotes and eukaryotes
rRNA is neither capped nor polyadenylated and is made in the nucleolus
The nucleolus:
Is significantly dedicated to the formation of ribosomal particles and is significantly enlarged during protein synthesis
Ribosomes - in Prokaryotes (E. coli):
- Various subunits and their ribosomal components are most easily isolated and characterized based on their sedimentation rate during centrifugation ( measured in S)
- S is not additive:
Monosome of bacteria = 70S (50S + 30S) – not additive
Large subunit: 23 S + 5 S rRNAs + 33 proteins
Small subunit: 16 S + 21 proteins
Ribosomes - in Eukaryotes (mammals):
- Larger than prok.
Monosome: 80 S particle (60S + 40S)
Large: 28 S + 5.8 S + 5 S + 47 proteins
Small: 18 S + 33 proteins
Components of pro- and eukaryotic ribosomes
- ribosome structure is fairly conserved in prok and euk.
ribosomes - Major differences are:
> in the size of the various different subunits
> Num. of proteins
> and the fact that there’s an additional rRNA in the larger
euk. ribosome subunit ( 5.8 S) - has no counterpart in
bacterial cell unit
tRNA
- Small (75-90 nucleotides), stable - easy to study - best
characterized of all RNA molecules - Structure is practically identical in prokaryotes and eukaryotes
- Transcribed from genes as larger precursors; but then get
spliced into mature 4S tRNA molecules
Holley et al. 1965:
- The first tRNA sequence was determined from a yeast tRNA
which carries alanine as its cognate amino acid
- established complete nucleotide sequence of tRNAala
- they identified a number of unique nucleotide bases which are
restricted to tRNAs and which have modified bases
> Post-transcriptional modifications as the modifications to
the bases happen after transcription
E.g. inosinic acid; ribothymidylic acid and pseudouridylic acid\
- tRNA transcription takes place using standard RNA bases
G/A/U/C - and the relevant bases are then modified - believed that these modified bases enhance hydrogen
bonding efficiency during the translation process
tRNA structure (2-dimensional)
Holley et al:
- It was already known that tRNA had a secondary structure caused by intra-strand base pairing
- used this to propose 2-dimensional cloverleaf model
- In this model, the arrangement of the linear sequence results in
base pairing which creates paired stems and unpaired loops
- The modified bases which didn’t form base pairs = loops
- Knowing GCU, GCC, GCA and GCG all encode alanine
- Holley looked for an anti-codon sequence which would be
complementary to one of these codons
- They found the sequence CGI (Anticodon 3’-5) in one of the
loops = anticodon loop
- Base I can form H bonds with U, C or A - which is the third
base in 3/4 of the alanine codons (wobble hypothesis)
> By having base I at position 1 in the tRNA - it corresponds to
the mRNA
> a single tRNA can uncover 3 of the mRNA codons
Consistent features:
- 4 large and 1 small stem:
- Anticodon stem with 7nt loop (central 3 = anticodon)
- 3’ end = Acceptor-stem with amino acid binding site
(pCpCpA-3’) - always has sequence CCA
- 5’-end: always has a G at the end of the molecule
Lengths of stems and loops very similar; anticodon loop is always found on same location on cloverleaf structure.
tRNA structure (3-dimensional)
[Rich et al.]
- 3-dimensional =L-shaped structure
- Anticodon loop and amino acid binding sites on opposite legs
of L
Charging tRNAs: aminoacylation
- Before translation can take place, the tRNA molecules must
be covalently linked to their cognate amino acids
= charging/ aminoacylation - tRNA in free or charged conformation
- Aminoacyl tRNA synthetases control charging of tRNA
- due to the “Wobble hypothesis” – minimum of 31 tRNAs
- But there are actually 20 different aminoacyl tRNA synthetases
- highly specific - one for each of the 20 amino acids -
regardless of the number of corresponding tRNAs - This is NB NB in maintaining the fidelity of translation
- transfer amino acids to 3’-A residues on tRNA’s.
Isoaccepting tRNAs:
Different tRNA’s which accept the same amino acid
Charging tRNAs: aminoacylation continued
- During the charging process, the amino acid is first activated by reacting it with ATP
–> forms an aminoacyladenylic acid complex; in which a
covalent linkage is made between the 5’ phosphate group
of AMP and the carboxyl end of the amino acid - at the same
time, pyrophosphate is released
Amino acid + ATP → aminoacyladenylic acid (activated) - This reaction takes place in conjunction with the enzyme
= an activated enzyme complex
[In association with aminoacyl tRNA synthetase] - This complex then reacts with a specific tRNA to transfer the
Amino Acid to the tRNA where it is covalently bonded to the 3’- adenine residue - whilst the AMP is released by the enzyme
- The charged Aminoacyl tRNA is now ready for translation
process
