Nabeel 20

Aya10/11/2011
Al-Nobani
Translation of mRNA
Basheer
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Genetics – Lecture 13
Wednesday, 10/11/2010
Done By: Aya Al-Nobani

Translation of mRNA
Today, we'll start talking about protein synthesis (translation of
mRNA) and Doctor Zyad will continue with this subject after Eid.

* The outline of the upcoming lectures:

1. The different requirements of protein synthesis.

2. Some characteristics of mRNA.

3. Structure of ribosomes and polysomes.

4. Polarity of protein synthesis.

5. Function of transfer RNA (acts as an adaptor).

6. Activation of amino acids which are the building blocks of

polypeptides.

7. Amino acyl tRNA synthetase (the enzyme that activates

amino acids and charge tRNA).

8. The genetic code.

9. Codon-anticodon interactions & structure.

10. Initiation and stop codons in prokaryotes vs. eukaryotes.

11. Reading frame.

12. Mutations affecting translation thus affecting the reading

frame.

Please refer to the objectives of the lectures in the slides and

make sure you understand each and every one of them.

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 * Now the lecture begins …

(Slide 4)
This is the structure of a eukaryotic mRNA that is ready for
translation, so it’s capped by a 7-methyl guanine and two
other methyl groups on the first and the second nucleotides.
Then, there’s the 5’ untranslated region which is important,
because sometimes it has sequences that control the
transcription and translation. After that, there's an important
signal for the initiation of translation in eukaryotes and
prokaryotes; the initiation codon (AUG); without this signal,
translation will not be initiated.

The translation process will continue until it faces another

signal which is the termination codon (UGA); (note: there are
3 different termination codons or subcodons); the function of
this codon is to stop the translation process of mRNA. Then it
has the 3’ untranslated region, and then the poly(A) signal
and the poly(A) tail.

So the region that will be translated is the one between the

initiation and the termination codons, meaning that amino
acids (the building blocks of protein) will be formed into a
polypeptide chain according to this sequence of nucleotides.

Remember that this is the third step of the dogma of molecular

biology (the first two were replication & transcription).

* The Tools of Translation:

1. mRNA
2. Ribosomes
3. tRNA
4. Amino acids as building blocks

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 5.Energy
So we are going to talk about these components in details:

Ribosomes (slide #5):

1. Prokaryotic ribosome
- Small subunit: 30S subunit = 16S rRNA + 21 proteins
- Large subunit: 50S subunit = 23S rRNA + 5S rRNA + 35
proteins

* The assembly of the two subunits forms the whole ribosome

(the 70S ribosome).

2. Eukaryotic ribosome
- Small subunit: 40S subunit = 18S rRNA + 33 proteins
- Large subunit: 60S subunit = 28S rRNA + 5S rRNA + 5.8S
rRNA + 49 proteins

* The assembly of the two subunits forms the whole ribosome

(the 80S ribosome).

So ribosomes are nucleoproteins; they are composed of RNA

and proteins. The number of the subunit followed by (S)
represents the density or the segmentation rate of each
subunit. Note that the assembly of the whole ribosome from the
two subunits is not an additive process, because it represents
density, segmentation, conformational change, and biochemical
processes, so it’s not simple math.

* The 16S rRNA in prokaryotes has a special importance, why?!

>> It binds to the Shine-Dalgarno sequence, if you know
about it!

* Steps of the Translation Process

1. Initiation: starts by the assembly of the small and the large
subunits to form the whole ribosome which will search for the
initiation codon (AUG) on the mRNA.

2. Elongation: in this stage, the genetic information in the

mRNA will be expressed as amino acids hooked to each other
by peptide bonds, in the 5’- 3’ direction.

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3. Termination: once the ribosome reaches the termination
signal (UGA), there will be dissociation of the two subunits, and
the newly synthesized polypeptide chain will be released.
* Note: the direction of translation is the same as the direction
of transcription and replication (5’-3’), and the direction of
translation according to the newly synthesized protein is from
the N terminal to the C terminal.

As you see in slide #6, many ribosomes can work on the same
mRNA at the same time, so you can see many stages of
translation on the same mRNA by different ribosomes, and this
is called polysomes: many ribosomes translating the same
message.

Q: Do all ribosomes produce the same protein on the

same mRNA?
Yes. It’s the same mRNA, so all the ribosomes working on it
must produce the same protein unless mutations occur to the
message. Keep in mind that alternative splicing produces
different mRNAs but each mRNA will be translated into one
protein.

tRNA (slide #7)

It’s very important to the translation process; it acts as an
adaptor molecule since each tRNA will bind to a specific amino
acid and to the codon on the mRNA, and so they will be the
mean that arrange amino acid according to the genetic code on
the mRNA, in order to form peptide bonds between them.

* Regions of tRNA
1. 3’ stem acceptor (which ends with CCA in all tRNA): via
this region, the amino acid binds to the adenosine (A)
molecule. Every amino acid has at least one tRNA, and
there is a specific enzyme to help the amino acid to bind
with its specific tRNA.

2. The anti-codon region which interacts with the codon on

the mRNA.

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The acceptor
region

Anti-codon
region
So, according to the codons on the mRNA, the needed amino
acid will bind to its specific tRNA via the adenosine nucleotide
at the 3’ end and this will be facilitated by the enzyme amino
acyl tRNA synthetase. This enzyme will lead to the activation
of the amino acid in order to be coupled with the tRNA. So the
amino acids must be first activated in order to be used as
building blocks for proteins.

Without the unique secondary and tertiary structures of the

tRNA, it will not be able to recognise the codon on the mRNA
nor its specific amino acid, nor will it be recognised by the tRNA
synthetase, so the secondary and tertiary structures of the
tRNA are very important for the proper function of the tRNA.

Q: How is tRNA synthesized?

tRNA is synthesized by transcription of specific genes (there are
specific genes for each type of RNA) using RNA polymerase. For
example: rRNA will be synthesized from one gene as one big
molecule, and then this big molecule is cleaved into pieces to
give different types of rRNA: like 5S, 16S, 23S or 28S.

As you know, the building blocks of proteins are amino acids. In

order for the amino acid to be used, it has to be activated; it
has to become acyl amino acid. This activation process serves
to make the amino acid suitable as a substrate for the enzyme
(tRNA synthetase). Also, it provides it with the needed energy
for binding with tRNA, so amino acyl tRNA synthetases are
enzymes that charge amino acids to their specific tRNAs
(couple them). Every amino acid has its specific amino acyl

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tRNA synthetase, so there will be at least 20 types of this
enzyme (and at least 20 types of tRNA) to activate the 20 types
of amino acids, but in reality there are more than 70 types of
tRNA molecules.

Q: Why does each amino acid require a different type of

the enzyme?
This is because of the different structures of amino acids, and
the specificity of the enzymes (binding only to a certain
substrate). Also, having one enzyme to activate more than one
amino acid may activate unneeded amino acids which will be a
waste of energy (not economical to the cell).

There can be several isoacceptor tRNAs for the same amino

acid, and that explains why we have more than 70 types of
tRNA with different sequences and even different anti-codons;
(in other words, there can be more than one (some have 3, 4,
5, 6) tRNA that can bind with the same amino acid and have
different anticodons), but all of them use the same amino acyl
tRNA synthetase.
So although we have more than one tRNA for the same
amino acid, they all use the same amino acyl tRNA
synthetase enzyme.

The difference between the different tRNAs that bind with the
same amino acid is mainly in the anti-codon region in one
nucleotide.

Each amino acyl tRNA synthetase binds to:

1. amino acid
2. ATP: which is required to activate the amino acid, and the
amino acid will keep that energy to help in the upcoming
steps of protein synthesis.
3. isoacceptor tRNAs

So these enzymes are molecules that could bind to different

substrates: amino acid, ATP and tRNA. And each substrate has
its own binding site on the enzyme so that it can catalyse the
activation and the coupling of the amino acid to the tRNA.

Q: Why is it called isoacceptor?

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Acceptor because it accepts the amino acids, and iso because
there can be more than one tRNA for each amino acid.

This is the activated amino acid; it has an adenine that came

from the ATP and it’s called adenylated amino acid.

And then after activation, the same enzyme will charge and
couple this activated amino acid with the tRNA at the 3’ end
acceptor side, releasing AMP. The resulting substrate will be
used as a building block to synthesize the polypeptide chain
(protein).

* The genetic code (slide 10)

The genetic code consists of 64 triplet codons (this 64 results
from 43) as 4 is the number of the types of nucleotide (A, T, C,
G) and 3 is the number of nucleotides in each codon. Note that
if the codon has a less number of nucleotides, the resulting
possible number of codons will not exceed 20 which is the
number of amino acids (we should have more than 20 codon,
one for each amino acid in addition to the initiation and the
termination codons).
All codons are used in protein synthesis. How is that possible?
Each amino acid is represented by more than one codon, and
this is called degeneracy: having more than one codon for the
same amino acid. Also, we have 3 termination codons
(subcodons: UAA, UAG, UGA); you have to memorise these
codons because if I give you a sequence of mRNA and ask you
what will be the polypeptide chain that could be synthesized
from the sequence, you must be able to recognise the
termination codon to know when to stop.

AUG is a very important codon; it codes for methionine. Also, it

is the initiation codon for any polypeptide chain. So, all
polypeptides start with Met (AUG), although it is used internally
to code for normal methionine in a polypeptide chain. So the
starting point will be from the first AUG in the mRNA and the
rest will code for normal methionine. In order for this to be
accomplished, the starting AUG has a specific tRNA (called

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initiator tRNA) and a specific tRNA synthetase. All the other
internal AUG are recognised by different tRNA (to code for
normal methionine) and different tRNA synthetase.

In prokaryotes, this methionine (the starting one) will be

modified by adding a formyl group to it, and its tRNA is called
f-met tRNA and the (f) stands for formyl.

When a prokaryote that is living in a host cell produces a

protein containing this formyl methionine, the host cell will
recognise it as a foreign body and attack it. And if the protein
does not contain the f-met, it will not be recognised by the host
cell and antibodies will not be formed. Because of that, all
proteins produced by prokaryotes start with this formylated
met.

Back to slide (10)

• 5 amino acids are specified by the first two nucleotides
only (explained later).
• 3 additional amino acids (Arg, Leu, and Ser) are specified
by six different codons, as we said there are some amino
acids that have one codon, other have 2, 3, 4, 5 and even
6 codons. Please refer to slide # 11.

Q: How will the tRNA recognise the amino acid?

By the codon and anti-codon interaction and with the help of
tRNA synthetase, as we said before, the enzyme will bind to the
amino acid and the tRNA, activate the amino acid and then bind
it to the tRNA which will interact via the anti-codon with the
codon of the mRNA.

* Rules concerning Codon-anticodon interactions:

1. Codon-anticodon base-pairing is antiparallel: which
means that the mRNA and the tRNA run in opposite directions
(one in the 5’-3’direction and the other in the 3’-5’direction).
* Remember that we always read the codon starting
from the 5’end. *

2. The third position in the codon is frequently

degenerate (from degeneracy)

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3. One tRNA can interact with more than one codon, and
this goes by wobble rules:
If the third nucleotide of the anticodon was C it can bind with G
or I (inosine)
A with U or I
G with C or U
U with A, G, or I
I with C, U, or A

Because of these rules, one tRNA can bind with more than one
codon. For example: one tRNA leucine can read two of the
leucine codons, but it will not recognise a codon of a different
amino acid, and this explains what we said before: codons of
the amino acid are recognised by the first 2 nucleotides. So the
first 2 nucleotides represent the original amino acid.

Note that the wobble rules deviate from the Watson-crick rules
of base pairing.

* Initiation in prokaryotes and eukaryotes

* Please refer to slide #14 in order to fully understand the
following paragraph:
In both the prokaryotes and eukaryotes, the first AUG will serve
as the starting point and will be recognised by searching by the
help of small subunit ribosomes (which is composed of protein
and RNA molecules with shine-dalgarno sequence) will base
pair with that AUG.

In the prokaryotes, any AUG codon with shine-dalgarno site can

act as an initiating point regardless of its position (internally or
not). So the mRNA is called polycistronic meaning that it can
produce more than one protein.

On the other hand, in the eukaryotes, only the first AUG

downstream of the 5’cap can be the initiating codon. Other
internal AUG cannot serve as an initiating point and code for
normal methionine. So the mRNA is called monocistronic
meaning that it can only produce a single polypeptide chain.

* Reading frames:
The reading frame starts with the initiating codon and goes by
every subsequent triplet to be read as a codon until reaching

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the termination codon, this is the definition of the reading
frame: the whole translated region from the starting till the
finishing codon, reading all codons.

The resulting polypeptide sequence depends on the codons

within the translated region, so it’ll start with “met” coded by
the initiating codon AUG.

Sometimes, mutations occur to the mRNA resulting in the

change of the reading frame. If a deletion or insertion of one
nucleotide occurs, it will result in changing the reading
sequence of triplet, so the amino acid sequence will also
change. Check the example in slide #15 (it’s a disease that will
be explained later).

And the effect of the change depends on the type of mutations:

• Missense mutations (e.g., AGC Ser to AGA Arg):
change one of the amino acids with another, the effect
of this depends on whether the new one is of the same
type as the old one, and the location of the changed
amino acid (important within the active site or not).

• Nonsense mutations (e.g., UGG Trp to UGA Stop): so

the translation will stop before the right place due to
formation of a new stop codon, and the resulting
polypeptide chain is called truncated (shortened)
polypeptide chain, shorter than the normal, for example
if the normal protein consist of 100 amino acid, the
truncated may have 20. And the rest of nucleotides
which were supposed to be translated will not be used.

• Read through, reverse terminator, or sense

mutations (e.g., UAA Stop to CAA Gln): change of a
stop codon into a normal codon and then the translation
will not stop where it’s supposed to, but continue
further on.

• Silent mutations (e.g., CUA Leu to CUG Leu) do not

affect translation, the changed nucleotide resulted in
another codon that codes for the same amino acid.

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* If the mutation was in 3 nucleotides (the whole codon was
inserted or deleted) or multiple triplets, one or more amino
acids will be added or deleted and the rest will not be affected.
And if the removed amino acid had an important function in the
active site, the protein would not be functional. Otherwise the
resulting protein would be functional or partially functional.

An example of a genetic disease resulting from such mutations

is the haemoglobin Wayne (3’ terminal frame-shift mutation)
which results from deleting of a single nucleotide (U) from the
normal sequence, and this will change the whole sequence of
the following amino acids and the resulting haemoglobin is not
properly functional. Refer to slide #16 for details on the causing
mutation.

* The END *

Sorry for any mistakes

Thanx to all my friends :D

Special thanx to Randa Zayed & Samah Abu
Omar

Aya Al Nobani

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