Transcript charged

The genetic code, the molecular mechanism
of translation and the synthesis of proteins
The information encoded in DNA is transcribed into RNA and finally
translated into the sequence of proteins.
The genetic unit coding for one single amino acid is a codon. One gene
codes for one proteins, one cistron for one polypeptide chain. As many
proteins consist of only one polypeptide chain, many genes have only
one cistron.
Decoding of the information present in mRNA is carried out by ribosomes
(rs). Amino acids used for the polypeptide synthesis are transported to
the ribosomes by tRNA molecules (charged tRNAs). Aminoacylation of
tRNAs is catalyzed by very specific aminoacyl-tRNA synthases.
The energy required for protein synthesis is supplied by macroerg ATP and
GTP molecules.
The genetic code
coding for 20 amino acids (+ start and stop) 4 nucleotides are not enough
using 2 letters the number of combinations is still too few (16)
3 letter combinations are plentiful: 64. How many are used?
Does the code have commas or isthecodecontinuous?
(ATGcGTCaGGTaTTC...)?
Is the code overlapping or not? (ATG TCA CAA)?
GTT AGC
Experiments with intercalating mutagens suggested a 3 letter code:
activity of a bacterial enzyme was lost after mutagen treatment
a second mutation in the same gene did not change the situation
some of the further mutagenized bacteria recovered (part of) the enzyme
activity
Deciphering the code
Artificial mRNAs (synthetic oligo-ribonucleotides)
conduct the synthesis of peptides:
Poly rU: phenylalanin,
poly rC: prolin, poly rA: lysine, poly rG: no product
(– inhibitory secondary structure)
poly rAC (1:1) threonine + histidine
poly rAC (2:1) asparagine, threonine, glutamine
Ribosomes bind to nitrocellulose filters (tRNAs do not)
aminoacyl-tRNAs (labelled with radioactive amino
acids) are attached to ribosomes in the presence of
ribo-trinucleotides. 64 different triplets yielded the
complete code library.
The code is made up of triplets, it is degenerated
(several codes can code for the same amino acid),it
is non-overlapping and comma free.
One of codons of methionine (ATG=AUG) serves as
start signal, but the stop codons code no amino
acids.
Degenerated code, codon usage
Certain amino acids are coded by several codes. It does not mean as many tRNAs. In
different organisms the preferred codon for the same amino acid can be different.
Proteins made in large quantities use preferred codes (red bars),
Most codes are universal, a few differences exist. The third letter of the code can
wobble.
There are 3 stop codons:
UGA, UAA, UAG.
In certain mutant bacteria
one of the stop codons
code for amino acid:
fusion proteins not
very frequent
(amber, ochre, opal)
Remember: the structure of tRNAs
tRNA is similar to a clover leaf. Three nucleotides of the blue anti-codon
loop are complementer with the codon in the mRNA. Other loops
and special bases serve as recognition and/or identification signals
for aminoacyl-tRNA synthases and translational factors.
Identification points on the tRNA molecule
Stuctural elements of the tRNA molecule also serve as identification points for
aminoacyl-tRNA synthases and elongation factors (EF-Tu). The figure shows
the most important identification points on the molecule (large spot:
important base). The special bases (T, pseudoU, D, psi, Y) also serve as
identification points.
The structure of the 16S rRNA
rRNA serves as a skeleton
for binding ribosomal
proteins, but it is also a
molecule with catalytic
activity
Assembly of the ribosome
The precursor of the ribosomal RNA is digested by and endonuclease
and then cut back by an exonuclease to produce mature rRNA.
Formation of RNA loops and binding of specific proteins leads to the
formation of the active ribosomal subunit.
Formation of a
ribosome
Both bacterial and eukaryotic
ribosomes have two subunits
of similar, but not identical
structure. There are effective
antibiotics based on the
differences of the pro- and
eukaryotic ribosomes.
The exact 3D structure of the rs
is not known but we have
detailed (X-ray crystallographic, electron microscopic,
biochemical) informations on
it.
Binding sites on the ribosome
E, P and A sites are tRNA binding sites. mRNA is bound to the small subunit.
Large subunit
of the ribosome
Anatomy of the ribosome
The large subunit has a
„tunnel” for the freshly
synthesized polypeptide
chain.
The ribosome reads the
mRNA from the 5’ end
and makes the polypeptide chain from the N
terminal end towards the
C terminal end.
Finding the start site
The mRNA has long untranslated ends on both sides. The start is
determined by the Shine-Dalgarno box (red): the following AUG
sequence codes for the first (always methionine) amino acid.
Initiation of the process
Binding of the large subunit
Elongation
The aminoacyl-tRNS enters site A
tRNA with an anticodon
complementer to the
codon enters the A site.
The large subunit shifts
right.
Trans-acylation
The peptide chain (as an acyl reagent) jumps from the OH group of the tRNA to the
NH2 group of
the incoming aminoacyl-tRNA. The free tRNA enters the E site,
the A site is liberated. The large subunit returns to its original position: to left.
The empty tRNA is rejected
The new aminoacyl-tRNA enters the A site
The peptide chain gets longer
Initiation factors of the synthesis
IF-4
Elongation factors
A number of protein factors help the work of the ribosomes and tRNAs.
During elongation EF-Tu
is one of the most important
participant.
EF-Tu is responsible for the
recognition and proper
positioning of tRNAs
Fig shows the structure of
the tRNA EF-Tu complex
The EF-Tu cycle
Function of EF-Tu
Highly productive synthesis
The protein, which binds the
polyA tail (polyA binding
protein I) has affinity to the
initiation factor IF-4, which
binds the cap of the mRNA.
As the two proteins interact, they
produce a circular mRNA,
bringing together the start
and stop codons. This
arrangement helps the
frequent initiation and highly
productive translation.
Synthesis of membrane proteins and secreted
proteins
Proteins with transmembrane domains, proteins which are produced for
export or proteins extensively modified in the lumen of the RER and the
Golgi must cross the membrane of the ER.
A signal sequence, composed of apolar amino acids is located at the N
teminal end of the protein. This is bound by the SRP (signal recognition
particle).
Signal sequence and SRP
SRP binds to the signal sequence, blocking translation, until it can interact
with its receptor on the surface of the ER membrane.
Then translation resumes,
signal peptide remains
bound in the pore,but the
growing peptide chain
enters the lumen.
Finally the signal peptidase
clips off the signal peptide.
Crossing the membrane of the ER
Signal peptide, SRP and its receptor