241 Biol - 2012
Lecture 19: Translation
Translation of mRNA, occurred at the ribosomes, is the biological polymerization of
amino acids into polypeptide chains.
I) Features of the genetic code
a) A codon, consisting of three ribonucleotides, specifies one amino acid. In 1960s,
Sydney Brenner argued on theoretical grounds that the code must be a triplet.
This was experimentally demonstrated by analyzing revertants of frameshift
mutations in the phage rII locus (Crick et al.).
b) The code is unambiguous. One codon specifies one single amino acid.
c) The code is degenerate. Some amino acids (18 of 20) are specified by more than
one codon.
d) The code contains start and stop.
e) The code is non-overlapping. If the code is overlapping, there will be restrictions
to the order of amino acids (S. Brenner). Furthermore, if the code is overlapping,
one mutation could affect multiple amino acids.
f) The code is nearly universal. Exceptions in mitochondria and some bacteria.
II) Deciphering of the genetic code a) They (Nirenberg and Matthaei, 1961) used an in vitro (cell-free) proteinsynthesizing system and the enzyme polynucleotide phosphorylase. The key
point is that the addition of a ribonucleotide by polynucleotide phosphorylase is
random, based on the relative concentration of the four NDPs. In another word,
the probability of the insertion of a specific ribonucleotide is proportional to the
availability of the molecule, relative to other available nucleotides.
b) Homopolymer RNAs consist of only one type of nucleotides (i.e. AAAA,
UUUU, CCCCC, or GGGGG) were added to several in vitro systems, each with a
different amino acid made radioactive. It was found that AAA codes, for lysine,
UUU codes for Phenylalanine, and CCC codes for proline. GGG did not work
because of some technical issues.
c) Mixed copolymer RNAs made from NDPs of different proportions were added
to the in vitro system. The percentage of particular amino acid (hot) incorporated
should be proportional to the frequency of a specific codon. Please make sure
that you can do this!
d) Triplet binding assay short mRNA and ribosomes serve as a binding site for
radioactive labeled charged tRNA. The anticodon in tRNA is complementary to
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the codon in mRNA.
e) Repeating copolymer (G. Khorana) synthesized RNA containing di- or trinucleotide repeats.
III) Wobble hypothesis (Crick) The initial two ribonucleotides of the triplet codes are
more critical than the third. The hydrogen bonding at the third position of the codonanticodon interacton would be less constrained.
I)
One gene one enzyme theory (Beadle and Tatutm): By isolating Neurospora
auxotrophic mutants, they showed that each can be rescued when supplied
with a specific Thus nutrient. mutations disrupt enzymes that are responsible
for synthesis of these nutrients.
II)
Ribosomes: Ribosomes facilitate the translation of mRNA into proteins.
Each ribosome monomer consists of large and small subunits, both of which
contain proteins and RNAs. The prokaryotic large subunit contains 23S RNA,
5 S RNA, and 31 ribosomal proteins. The prokaryotic small subunit contains
16S RNA and 21 proteins. The RNA components of ribosome appear to be
critical for ribosomal function. The genes for rRNA are presented in multiple
copies in the genome (ie repetitive DNA).
III)
tRNA: composed of 75 to 90 nucleotides and displays the cloverleaf
structures with 3 overhang. Through post-transcriptional modifications,
tRNAs contain unusual bases (therefore unusual base-pairing properties). For
instance, in the anticodon loop of tRNAala, CGI (3 to 5 direction was found.
The nitrogenous base I (inosinic acid) can form hydrogen bonds with U, C, or
A at the third position of the codon triplets.
IV)
tRNA charging: Before translation can proceed, tRNAs need to be
chemically linked to their respective amino acids. This process is called
charging (or aminoacylation) and is mediated by aminoacyl tRNA
synthetase.
V)
Translation:
a) Initiation: In E. coli, translation initiation involves the small ribosomal subunit,
an mRNA, a specific charged initiator tRNA, GTP, Mg2+, initiation factors (IFs).
Initiation factors are released from ribosomes once initiation is completed. The
small ribosomal subunit, along with the IFs, binds mRNA at the Shine-Dalgarno
sequence, which precedes the AUG start. At the AUG start, charged
formylmethionyl tRNA binds and sets the reading frame. Then the ribosomal
large unit binds to this initiation complex, and the initiation factors are released.
b) Elongation: Once both subunits of ribosomes are assembled, binding sites for two
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charged tRNAs are formed. They are designed as the P (peptidyl) and the A
(aminocyl) sites. When both sites are occupied, peptidyl transferase catalyzes the
formation of the peptide bond that links the two amino acids together. The
covalent bond between the amino acid the tRNA occupying the P-site is
hydrolyzed. The resulting dipeptide is attached to the 3end of the tRNA in the A
site. The uncharged tRNA then transiently moves to the E (exit) site and is
released from the large ribosomal subunit. The mRNA-tRNA-peptide complex
then shifts in the direction of the P site.
c) Termination: The stop codon signals the action of GTP-dependent release
factors, which cleave the polypeptide chain from the terminal tRNA, releasing it
from the translation complex. Then tRNA is released from the ribosome, which
dissociates into subunits.
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