Slajd 1

[MCB 411/411H
Module 17
The Mechanism of Translation]

An overview of translation

In order for proteins to be made in cells, the
following components are necessary:

-messenger RNA (mRNA)

-ribosomes (complexes of protein and
ribosomal RNA [rRNA])

-transfer RNA (tRNA)

The process of translation has four

stages:

1.tRNA charging (adding the correct amino

acid to the correct tRNA)

2. initiation (assembly mRNA, ribosomes, and

tRNA)

3. elongation (reading the code and

converting the information into a peptide)

4. termination (ending the synthesis of a

protein)

The general structure shown on the left has the

important features that you will find in all tRNAs.
This includes the base paired stems, including the
acceptor stem, and the loops (the D-loop, the TYC-
loop and the anticodon loop). D and Y are symbols
that indicate modified bases that are present in
tRNAs and not generally found in other RNAs.

These modified bases are shown in Figure:

The significance of the modified bases is not
totally clear. The modifications take place after
the transcription of the tRNA from the DNA
template. Data suggest that if the bases are left
unmodified the tRNA does not function properly.
What their exact role is remains unknown.

Of course the cloverleaf structure is just a cartoon representation.
Another representation of the molecule comes from X-ray
crystallography. In this case, it appears that the molecule is folded up
into the form shown in Figure

The  various  regions  of  the  molecule  are  color
coded as in Figure.
This  folded  structure  is  thought  to  be  more
representative  of  the  molecule  as  it  might
occur  within  the  cell.  These  models  of  tRNAs
are  quite  striking  in  three  dimensional
representation:

Transfer RNA Charging

The process of adding the correct amino acid to
the  tRNA  responsible  for  bringing  to  the
ribosome  is  called  "charging."  The  word
"charge"  here  is  used  in  the  sense  of  "loading,"
as in charging a battery.

The charging reaction is catalyzed by a set of
enzymes called aminoacyl tRNA synthetases.

The enzyme catalyzes a two-step reaction:

1.In the first step, utilizes ATP and the amino

acid is energized by the covalent addition of
AMP, joining the two molecules by making a
bond between the phosphate of AMP and the
COOH of the amino acid.

This splits of the pyrophosphate of ATP,
which is immediate broken down into to
inorganic

phosphates

the

enzyme

pyrophosphatase.

Therefore, the energy of two phosphodiester
bonds drives this reaction.

2. The second step is the transfer of the
amino acid to the 3' or 2' OH of the terminal A
of the tRNA.

The product of the reaction is an aminoacyl-
tRNA and AMP.

The overall reaction is as follows:

amino acid (aa) + ATP + tRNA --> aminoacyl-
tRNA + AMP + 2 Pi

The link to the tRNA can be initially at either
the 3' OH or the 2' OH.

The 3' end of every tRNA is the sequence
---CCAOH.

The specificity of
these enzymes
(aminoacyl tRNA
synthetases) is
determined by the
structure of the
tRNA.

Although you would
think that the
anticodon provides
all of the information,
an analysis of various
tRNA structures
shows that the
important bases
might be at other
locations in the
molecule.

Prokaryotic Initiation

Protein synthesis in all cells begins (is initiated)
by the association of mRNA, tRNA and
ribosomes.

This initiation is mediated by a series of factors
appropriately called initiation factors (IFs).

In Prokaryotes there are three: IF-1, IF-2, and
IF-3.

The first step is to make sure that the ribosome
is dissociated into it's subunits.

This is accomplished by two of these factors.

This sequence of events is diagrammed in Figure:

In  the  cell  intact  ribosomes  (70S  ribosomes)
interact with IF-1 to cause dissociation into the
two  subunits,  30S  (the  small  subunit)  and  50S
(the  large  subunit).  IF-3  then  binds  to  the  30S
subunit  and  keeps  the  two  from  reassociating
until the time is correct.

The  first  event  of  interaction  between  the
components  of  translation  is  the  formation  of
the  30S  initiation  complex.  This  involves  the
30S  subunit,  the  mRNA,  and  a  special  initiator
tRNA.

The code word that says "start protein
synthesis" is AUG, the same code word that
means "methionine".

As you might expect, the tRNA that is the
initiator will be one that can read this code
word.

However, in prokaryotes, this special tRNA
carries a modified version of the amino acid
methionine.

Figure shows the structure of methionine and
N-formyl-methionine:

In prokaryotes, the 30S subunit of the ribosome,
along with fMet-tRNAMetf, interacts directly with
the  start  codon  (AUG)  that  begins  the  protein  to
be translated. All three initiation factors (IF-1, IF-
2,  IF-3)  are  involved  in  this  process.  IF-2  carries
out  it's  function  in  this  event  by  having  a  GTP
bound  to  it.  The  energy  from  this  GTP  will  be
used a bit later.

The  precise  placement  of  the  30S  subunit  at
the  start  codon  is  achieved  by  a  base  pairing
between the 3' end if the 16S rRNA of the 30S
subunit  and  sequences  found  just  upstream
from the initial AUG.

This  base-pairing  interaction  was  discovered
by  John  Shine  and  Lynn  Dalgarno  and  the
sequence  in  the  mRNA  is  therefore  called  the
Shine-Dalgarno sequence (SD sequence).

This base-pairing is shown in the following
figure:

Once the 30S subunit is located correctly, the
initiator tRNA is positioned over the AUG codon
that begins the protein.

This forms the 30S initiation complex.

The SD-sequence explains how prokaryotic
mRNA can be polycistronic.

Since the 30S subunit finds its way to each
start codon, several different starts can be
made on the same mRNA, if they have the SD-
sequence upstream.

Here's the lac messenger RNA as an example,
showing the location of the SD-seqences
upstream of each gene:

Next, the 50S subunit joins this complex, to
form the 70S initiation complex.

The three factors now leave and the GTP that
is attached to IF-2 is hydrolyzed to GDP and
Pi.

All of the events of prokaryotic initiation are
summarized in the following table and in
Figure:

Steps in Formation of

70S initiation

Complex:

1. dissociation of the

70S ribosome into the

30S and 50S subunits

(IF-1)
2. binding of IF-3 to 30S
3. binding of IF-1 and

IF-2/GTP to 30S
4. joining of 30S/IF's

with initiator tRNA and

mRNA to form 30S

initiation complex
5. Binding of 50S to 30S

complex, with loss of IF-

1 and IF-3
6. dissociation of IF-2

with hydrolysis of GTP

to GDP and Pi

Eukaryotic Initiation

The beginning of protein synthesis in
eukaryotes also involves the association of
ribosome, tRNA, and mRNA.
However, unlike the prokaryotes, this
association takes place in a very different way.

Remember that the 5' end of eukaryotic
mRNAs has a methylated cap.
Marilyn Kozak discovered that eukaryotic
ribosomes do not bind directly to the region of
the messenger RNA that contains the AUG
initiation codon.

Instead, the 40S ribosomal subunit starts at
the 5' end and "scans" down the message until
it arrives at the AUG codon.

This is diagrammed in Figure:

The AUG that is recognized likely sits within
a certain sequence context with the
following consensus:

CCRCCAUGG

where R is a purine (A or G).

In addition to the 40S subunit, this requires

energy (GTP), eukaryotic initiation factors (eIFs,
see below) and an initiator tRNA.

Unlike the prokaryotic case, the methionine
carried by this tRNA is not formlyated. The tRNA
is called tRNAi.

When it carries methionine, it is called Met-
tRNAiMet.

The formation of the initiation complex in
eukaryotes proceeds as outline in Figure:

The  40S  subunit  joins  with  eIF-3  to  form  the
40SN structure that can join with the initiator
tRNA.
Along  with  eIF-2,  the  tRNA  joins  to  form  the
43S complex.
This now binds to the capped 5' end of mRNA,
under  the  direction  if  eIF-4,  and  scans  down
the  mRNA  until  it  reaches  the  correct  AUG
start codon.

This is the 48S initiation complex.

The 60S subunit now joins, using the action of
eIF-5, to form the final 80S initiation complex.

The unique event in this sequence that truly
distinguishes this from prokaryotic initiation is
the scanning of the 40S subunit.

This requires the presence of the 5'
methylated cap structure.

The initiation factor responsible for this
recognition is a factor called eIF-4F.

This consists of three separate proteins:

-eIF-4E (the cap binding protein itself),
-eIF-4A and
-eIF-4G.

All three together are eIF-4F.

The best evidence that the methylated cap is a
necessity for translation comes from infection of
cells with poliovirus.

This virus has an RNA genome that serves as a
messenger RNA inside the cell.

The genomic RNA of poliovirus is not capped
and methylated, and yet is translated in the
infected cell very efficiently.

It turns out that shortly after infection,
poliovirus shuts down the cell's ability to
translate any capped mRNA.

It does this by destroying eIF-4G, the stabilizing
protein of the cap binding complex eIF-4F.

How  is  poliovirus  RNA  translated  if  it  has  no
cap?  The  viral  RNA  has  an  internal  ribosome
entry site (IRES), a secondary structure feature
of the RNA that allows direct ribosome binding,
similar  to  that  found  in  prokaryotes.  Here's  a
diagram of the poliovirus IRES:

This feature becomes the place where the 40S
complex  binds.  The  Figure  is  supposed  to
show  the  situation  in  poliovirus-infected  cell,
with a protein factor called "X" binding to the
IRES.
There  is  clear  evidence  in  the  literature  that
documents the existence of this protein.

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