Protein Synthesis

Fundamental reaction:
the formation of a peptide bond btw the carboxyl group of the growing
polypeptide and a free amino group on an incoming aa .
Protein is synthesized from the N to C terminus .

Protein synthesis is performed by the ribosome a catalytic machine

with over 50 different proteins and many RNA molecules, rRNAs.
Millions of ribosomal subunits are assembled in the nucleolus. Two
ribosomal subunits are exported to cytosol where they join on an
mRNA to synthesize a protein.
Small subunit: provides the framework on which the tRNAs
accurately match to the codons on the mRNA;
Large subunit: catalyzed the formation of the peptide bonds that link
the aa together.

When not synthesizing protein, the subunits are separate.

They join on the mRNA molecule near its 5end.
The mRNA is then pulled through the ribosome; as codons enter
the ribosome, the mRNA is translated using tRNAs as adaptors;
when a stop is encountered the ribosome releases the finished
protein and the two subunits separate.
This is a fast event, 2 aas are added per second (2 Hz).
Ribosomes contain 4 binding sites,
one for mRNA
three, (E, P, and A sites) for tRNAs.
A tRNA is held tightly at the A and P sites only if its anticodon matches a
complementary sequence on the mRNA.
The A and P sites are close enough that two tRNAs are forced to form base
pairs with adjacent codons. This is a proofreading feature.

A-aminoacycl-tRNA
P-Peptidyl-tRNA
E- exit

Translation Initiation
1. tRNA-methionine is loaded on small subunit of

ribosome with eukaryotic initiation factors or eIFs .

It is loaded specifically with the help of eIF2. The initiator
tRNA can bind the small subunit without the complete
ribosome being present and binds directly to the P site.

2. The cap-structure at the 5end of the messenger

RNA is recognized by initiation factor eIF4F.

3. Translation begins with the AUG or start codon
and a special tRNA is required.
This initiator tRNA always carries the methionine aa.
All newly made proteins have M as their first aa but later
have it removed with a specific protease.

1.Formation of a 43S preinitiation complex comprising of

a 40S subunit, eIF1, eIF1A, eIF3, eIF2GTPMet-tRNA
(ternary complex) and probably eIF5
2.mRNA activation, during which the mRNA capproximal region is unwound in an ATP-dependent
manner by eIF4F with eIF4B
3.Attachment of the 43S complex to this mRNA region
4.Scanning of the 5 UTR in a 5 to 3 direction by 43S
complexes

5. Recognition of the initiation codon and 48S initiation complex

formation, which switches the scanning complex to a closed

conformation and leads to displacement of eIF1 to allow eIF5mediated hydrolysis of eIF2-bound GTP and P release
6. Joining of 60S subunits to 48S complexes and concomitant

displacement of eIF2GDP and other factors (eIF1, eIF3, eIF4B,

eIF4F and eIF5) mediated by eIF5B
7. GTP hydrolysis by eIF5B and release of eIF1A and GDP-bound eIF5B

from assembled elongation competent 80S ribosomes

8. Translation is a cyclical process, in which termination follows

elongation and leads to recycling, which generates separated

ribosomal subunits.

eIF4F is a complex of three polypeptides:

(a) eIF4A, an RNA-dependent ATPase and RNA helicase;
(b) eIF4E, a 24-kDa cap-binding protein; and
(c) eIF4G, a large polypeptide containing binding sites for

eIF4F interacts with both the cap (through eIF4E) and the
ribosome- associated eIF3 (through eIF4G).
Thus, eIF4F executes the pivotal function of bridging the
mRNA and the ribosome.
This type of initiation is known as cap-dependent initiation
and is the most common type of initiation. Initiation can
also occur through an alternative cap-independent initiation
mechanism, such as IRES (internal ribosome entry site).

New aa is added to poly peptide:

tRNA binding,
peptide bond formation,
large subunit and small unit translocation.
Entire ribosome moves three nucleotides
along the mRNA due to translocation
steps.

Step 1: tRNA carrying next aa, binds to

the A site by base pairing, so that the P
and A contain adjacent bound tRNAs.
Step 2: carboxyl end of the polypeptide
chain is released from the tRNA at the P
site and joined to the free amino group of
the aa linked to the tRNA at the A site,
forming a new peptide bond. This is
catalyzed by peptidyl transferase in large
subunit.
Step 3: large subunit moves relative to
the mRNA, shifting the two tRNAs to the
E and P positions
Step 4: small subunit and mRNA move
three nucleotides reseting the ribosome
to receive another tRNA.
This four step process is repeated for
each aa to be added.

Elongation factors:

EF-Tu and EF-G in bacteria or EF1 and EF2 in eukaryotes, enter

and leave the ribosome during each cycle
they help move translation forward and check if tRNA-aa match
is correct.
They monitor the interaction btw anticodon and codon make
sure this is accurate.

Ribosome as Ribozyme:

Ribosome is 2/3 RNA and 1/3 protein.

rRNA is on surface while protein in the interior.
Metal ions, often important when RNA is used to catalyze
reactions, not necessary for ribosome function. Believed that
23S rRNA forms a highly structured pocket that orients the two
reactants, peptide chain and tRNA, and greatly accelerates the
process.

RNAs with catalytic activity are called ribozymes.

The stop site

Stop Codons:

Stop codons, UAA, UAG, UGA, not recognized by tRNA and dont
specify an aa but signal ribosome to stop translation.

Release factors:

bind to any ribosome with a stop codon in the A site

force the peptidyl transferase to add a water molecule instead of an
aa to the peptidyl-RNA.
This reaction frees the carboxyl end of the growing protein from the
tRNA and releases it into the cytosol.
The ribosome then releases the mRNA and separates into small
and large subunit.

Release factors mimic tRNA shape and charge to enter A

site.
mRNA are often translated within polysomes, large
cytoplasmic assemblies of several ribosomes as close as
80 nt on a single mRNA. Allow the cell to make many more
proteins within a given time frame.

Accuracy of translation requires a lot of energy.

Protein synthesis consumes more energy than
any other biosynthetic process.
At least 4 high energy phosphate bonds are split
to make each peptide bond,
two in charging a tRNA with an aa, and
two more in the ribosome itself.
If there are errors, and they have to be fixed, more
energy is consumed.

While a protein is being synthesized it must be folded properly or

inappropriate aggregation might occur.
Molecular chaparones help these proteins refold, or
Chaperonins, can form an isolation chamber around the misfolded
protein to prevent aggregation until an environment is reached when
they can refold or be degraded in a proteosome.
The proteosome is an abundant ATP-dependent protease that is 1%
of all protein.
It consists of a central hollow cylinder, whose active sites face the
cylinders inner chamber.
Target proteins are threaded through the core, where they are
degraded; driven by ATP hydrolysis, proteins are unfolded exposing
them to the proteases lining the core; the unfoldases are called AAA
proteins.

This is a complex machine, the 19S cap acts as a gate, binding to

certain proteins to the proteosome, and it works until the protein is
broken up in to short peptides. Proteosome works on proteins
marked by small protein ubiquitin. Ubiquitin can mark some
proteins for descruction but can also be used for other signaling.
The number of ubiquitin molecules determine this.