Molecular Biology (CLS 354 )

DNA , replication, transcription, translation -2

Dr/ Abdul-Raouf

Translation - Ribosomes
Two substances that play key role in translation:
ribosomsS & transfer RNA
Ribosomes are the protein synthesizing machines
Ribosome subunits are designated with
numbers such as 50S or 30S.
Number is the sedimentation coefficient - a
measure of speed with which the particles
sediment through a solution spun in an
ultracentrifuge
Each ribosomal subunit contains RNA and protein.
The two ribosomal subunits both contain
ribosomal RNA (rRNA) molecules and a variety of
proteins
rRNAs participate in protein synthesis but do NOT
code for proteins
No translation of rRNA occurs

(a)

(b)

Figure shows E. coli ribosome structure.

(a) The 70S ribosome is shown from the side with the 30S particle (yellow) and the 50S
particle (red) fitting together.
(b) The 70S ribosome is shown rotated 90 degrees relative to the view in part (a). The 30S
particle (yellow) is in front, with the 50S particle (red) behind.

Summary
Ribosomes are the
cells protein factories
Bacteria contain 70S
ribosomes
Each ribosome has 2
subunits
50 S & 30 S
Each subunit contains
rRNA and many
proteins
Figure shows Composition of the E. coli ribosome.
The arrows at the top denote the dissociation of the 70S ribosome into
its two subunits when magnesium ions are withdrawn. The lower arrows
show the dissociation of each subunit into RNA and protein
components in response to the protein denaturant, urea. The masses
(Mr, in daltons) of the ribosome and its components are given in
parentheses.

Translation Adapter Molecule

Generating protein from ribosomes requires change from the
nucleic acid to amino acid
This change is described as translation from the nucleic acid
base pair language to the amino acid language
Crick proposed that some type of adapter molecule was needed
to provide the bridge for translation, perhaps a small RNA

Transfer RNA: Adapter Molecule

Transfer RNA is a small RNA that recognizes both RNA and amino
acids
A cloverleaf model is used to illustrate tRNA function
One end (top) binds amino acid with sequence specific to a
particular amino acid
Bottom end contains a 3 base pair sequence that pairs with
complementary 3-bp sequence in mRNA

Figure 3.17 Cloverleaf structure of yeast

tRNAPhe.
At top is the acceptor stem (red), where the
amino acid binds to the 3-terminal
adenosine.
At left is the dihydro U loop (D-loop, blue),
which contains at least one dihydrouracil
base.
At bottom is the anticodon loop
(green), containing the anticodon.
The T-loop (right, gray) contains the
virtually invariant sequence TC.
Each loop is defined by a base paired
stem of the same color.

Codons and Anticodons

Enzymes that catalyze attachment of amino acid to tRNA are
aminoacyl-tRNA synthetases
A triplet in mRNA is called a codon
The complementary sequence to a codon found in a tRNA is
an anticodon.
Definition :
Codon : Three-base segment of mRNA that specifies amino acids.
Anticodon:
1. Three-base segment of tRNA that docks with a codon.
2. Docking results in deposition of amino acid.

Figure shows Codonanticodon recognition.

The recognition between a codon in an mRNA and a
corresponding anticodon in a tRNA obeys essentially
the same WatsonCrick rules as apply to other
polynucelotides.
Here, a 3AAGm5 anticodon (blue) on a tRNAPhe is
recognizing a 5UUC3 codon (red) for phenylalanine
in an mRNA. The Gm denotes a methylated G, which
base-pairs like an ordinary G.
Notice that the tRNA is pictured backwards (3 5)
rela ve to normal conven on, which is 5 3, le
to right. That was done to put its anticodon in the
proper orienta on (3 5, le to right) to base-pair
with the codon, shown conven onally reading 5
3, left to right.
Remember that the two strands of DNA are
antiparallel; this applies to any double-stranded
polynucleotide, including one as small as the
3-bp codonanticodon pair.

Initiation of Protein Synthesis

The initiation codon (AUG) interacts with a special aminoacyltRNA
In eukaryotes this is methionyl-tRNA
In bacteria it is a derivative called N-formylmethionyl-tRNA
Position of the AUG codon:
At start of message AUG is initiator
In middle of message AUG is regular methionine
Shine-Dalgarno sequence lies just upstream of the AUG,
functions to attract ribosomes
Unique to bacteria
Eukaryotes have special cap on 5-end of mRNA

Translation Elongation
After initiation, initiating aminoacyl-tRNA binds to a site on the
ribosome, P site
Elongation adds amino acids one at a time to the initiating
amino acid
First elongation step is binding second aminoacyl-tRNA to
another site on the ribosome, A site
This process requires:
An elongation factor, EF-Tu
Energy from GTP

Figure shows Summary of translation

elongation.
(a) EF-Tu, with help from GTP, transfers the
second aminoacyl-tRNA to the A site. (The P
and A sites are conventionally represented on
the left and right halves of the ribosome, as
indicated at the top.)
(b) Peptidyl transferase, an integral part of
the large rRNA in the 50S subunit, forms a
peptide bond between fMet and the second
aminoacyl-tRNA. This creates a dipeptidyl-tRNA
in the A site.
(c) EF-G, with help from GTP, translocates the
mRNA one codons length through the ribosome.
This brings codon 2, along with the
peptidyl-tRNA to the P site, and codon 3 to the A
site. It also moves the deacylated tRNA out of the
P site into the E site (not shown), from which it
is ejected. The A site is now ready to accept
another aminoacyl-tRNA to begin another round
of elongation.

Termination of Translation and mRNA Structure

Three different codons (UAG, UAA, UGA) cause translation
termination
Proteins called release factors recognize these stop codons causing
Translation to stop
Release of the polypeptide chain
Initiation codon and termination codon at the ends define an open
reading frame (ORF)
SUMMARY Translation elongation involves three steps:
(1) transfer of the second aminoacy-tRNA to theA site;
(2) formation of a peptide bond between the first amino acid in the
site and the second aminoacyl tRNA in the A site;
(3) translocation of the mRNA one codons length through the
ribosome, bringing the newly formed peptidyl-tRNA to the P site.

Mutations
Genes accumulate changes or mutations
Mutation is essential for evolution
If a nucleotide in a gene changes, likely a
corresponding change will occur in an
amino acid of that genes protein
product
If a mutation results in a different
codon for the same amino acid it is a
silent mutation
Often a new amino acid is
structurally similar to the old and the
change is conservative

Sickle Cell Disease

Sickle cell disease is a genetic disorder
The disease results from a single base change in the gene for
b-globin
Altered base causes insertion an incorrect amino acid into
one position of the b-globin protein
Altered protein results in distortion of red blood cells
under low-oxygen conditions
This disease illustrates that a change in a gene can cause
corresponding change in the protein product of the gene