Central Dogma

Genetic information, stored in chromosomes and

transmitted to daughter cells via DNA replication is

expressed through transcription to RNA and

subsequent translation into polypeptide chains.

Central dogma: the flow of information from DNA to

RNA to protein.

Genetic Code

It is a dictionary that express the relation between

a sequence of nucleotides and a sequence of AAs.

Each individual word (codon) is composed of three

nucleotide and indicates an AA in the protein.

Codons are presented in mRNA language as

adenine (A), guanine (G), cytosine (C) & uracil (U).

Since 4 nucleotide bases are used to produce 3-base codons; that is (43) → 64 different

combinations of bases.

Term = chain terminator

codon.

AUG, codes for Met &

serves as the initiator

codon and also encodes

for internal Met in a

protein.

In mammalian

mitochondria,

AUA codes for Met

UGA for Trp

AGA and AGG serve as

chain terminators.

Genetic Code

Most AAs are coded by more than one codon?

Of the 64 codons, 61 code for the 20 AAs involved

in protein structure Val has 4 codons (GUU, GUG, GUC, GUA)

Phe has 2 codons (UUU, UUC)

Met has only one codon (AUG).

Termination (stop or nonsense) codons:

The 3 codons UAG, UGA, and UAA do not code for

amino acids, but are termination codons.

When one of these codons appears in an mRNA, it

signals that synthesis of the peptide chain coded for

by that mRNA is completed.

Degeneracy of codon

Consequences of altering the

nucleotide sequence

Point mutation: changing a single nucleotide base on the mRNA chain → one of three possible results

1. Silent mutation: The codon

containing the changed base

codes for the same amino acid.

2. Missense mutation: The codon

containing the changed base codes

for a different amino acid.

3. Nonsense mutation: The codon

containing the changed base

becomes a termination codon.

Possible effects of point mutations

Characteristics of genetic code

1- Specificity:

a specific codon always codes for the same AA.

2- Universality:

In all living organisms the genetic code is the same.

Exceptions???

3- Redundancy (degeneracy):

A given AA may have more than one triplet codon,

e.g. Arg & Leu each have six different codons.

4. Nonoverlapping and commaless:

the code is read from a fixed starting point as a

continuous sequence of bases, taken three at a time.

Reading frame

If one or two nucleotides

are either deleted from

or added to the interior of a message sequence →

frame- shift mutation

and the reading frame is

altered.

The resulting AA

sequence become

radically different from

this point.

Frame shift mutation

Components Required For Translation

(Protein Synthesis)

1. All the AAs that are found in the finished product.

2. The mRNA to be translated.

3. tRNAs.

4. Functional ribosomes.

5. Energy sources.

6. Enzymes & protein factors needed for the initiation,

elongation & termination of the polypeptide chain.

These gather in cytosol, like raw materials are

brought together in a factory, prior to turning on the

assembly line that creates a finished product.

Amino acids

All the AAs that eventually appear in the finished

protein must be present at the time of protein

synthesis.

If one AA is missing (e.g. the diet does not contain

an essential AA) that amino acid is therefore in

limited supply in the cell and translation stops at

the codon specifying that amino acid.

This demonstrates the importance of having all

essential AAs in sufficient quantities in the diet to

insure continued protein synthesis

Transfer RNA (tRNA)

At least one specific tRNA molecule is required per

AA.

In humans there are at least 50 different tRNA.

Because there are only 20 different AAs, some AAs

have more than one specific tRNA molecule.

This is particularly true of those amino acids that are

coded for by several codons.

1. Amino acid attachment site:

Each tRNA has an attachment site for a specific AA

at its 3'- end (-CCA-amino acid).

When a tRNA has a covalently attached AA it is said

to be charged.

when tRNA is not bound to an AA it is described as

being uncharged.

The AA that is attached to the tRNA molecule is said

to be activated.

2. Anticodon:

Three-base nucleotide

sequence that recognizes a

specific codon on mRNA.

This codon specifies the

insertion of the AA carried

by that tRNA into the

growing peptide chain .

tRNAs are known as

adaptor molecules??

Because they carry a

specific AA and recognize

the codon for that AA

Family of enzymes that attach AAs

to their corresponding tRNAs.

Each enzyme recognizes a specific

L-AA & the corresponding tRNA.

The enzyme catalyzes a two-step reaction → covalent attachment of

AA to the 3'-end of corresponding

tRNA.

The extreme specificity of the

synthetase in recognizing these

two structures is mainly

responsible for the high fidelity of

translation of the genetic message.

Aminoacyl-tRNA synthetases

Ribosomes are large complexes of

protein and rRNA.

They consist of two subunits-

one «large» and one «small» whose

relative sizes are given in terms of

their sedimentation coefficients,

or S (Svedberg) values.

Pro- and eukaryotic ribosomes are

similar in structure, and serve the

same function i.e. factories for the

synthesis of proteins.

Functionally competent

ribosomes

Protein Factors

1. Initiation,

2. Elongation

3. Termination (or release)

Some of these protein factors perform a

catalytic function,

whereas others stabilize the synthetic

machinery.

Sources of energy

ATP and GTP

Cleavage of four high-energy bonds is required for

adding one AA to the growing polypeptide chain:

A. Two from one ATP in aminoacyl-tRNA

synthetase reaction (one in removing PPi, and

one in the subsequent hydrolysis of the PPi to

inorganic phosphate by pyrophosphatase)

B. Two from two GTPs (one for binding the

aminoacyl-tRNA to the A site, and one for the

translocation step).

Codon Recognition by tRNA

Recognition of a particular codon in mRNA is

achieved by the anticodon sequence of the tRNA.

Some tRNAs can recognize more than one codon for a given amino acid.

A. Antiparallel binding between codon and

anticodon

B. "Wobble" hypothesis

A. Antiparallel binding between

codon and anticodon

Binding of tRNA anticodon to mRNA codon

follows the rules of complementary and antiparallel

binding:

the mRNA codon is read 5'→3' by an anticodon

pairing in the flipped (3'→5’) orientation.

When writing the sequences of both codons and

anticodons, the nucleotide sequence must

ALWAYS be listed in the 5'→3' order.

B. "Wobble" hypothesis

It describes mechanism by which

tRNAs can recognize more than

one codon for a specific AA.

The base at the 5'-end of the

anticodon (the «first base of the

anticodon) is not as spatially

defined as the other two bases.

Movement of that first base allows

nontraditional base pairing with the

3'-base of the codon (the «last»

base of the codon).

B. "Wobble" hypothesis

This movement is called «wobble» and allows

a single tRNA to recognize more than one

codon.

The result of this wobbling is ‘that there need

not be 61 different tRNA in order to read the

61 codons coding, for amino acids.

Steps in Protein Synthesis

Protein synthesis translates 3-letter alphabet

of nucleotide sequences on mRNA → 20-

letter alphabet of AAs.

mRNA is translated from its 5'- to 3'-end,

producing a protein synthesized from its

amino-terminal (N-terminal) to its

carboxyl-terminal (C-terminal).

Eukaryotic protein synthesis resembles that

of prokaryotes in most aspects.

Steps in Protein Synthesis

Each eukaryotic mRNA has only one coding

region: it is monocistronic.

In contrast, prokaryotic mRNAs often have

several coding regions: they are

polycistronic . Each coding region has its

own initiation and termination codon and

produces a separate type of polypeptide.

Translation is divided into 3 separate steps:

a- initiation. b- elongation. c- termination.

A) Initiation

It involves assembly of components of translation

system before peptide bond formation occurs. ???

1. two ribosomal subunits,

2. the mRNA to be translated,

3. the Met-tRNA

4. GTP (which provides energy for the process),

5. Initiation factors that facilitate the assembly of this

initiation complex

3.In prokaryotes, there are 3 initiation factors

(IF-1, IF-2, and IF-3), whereas in eukaryotes,

there are over ten (eIF to indicate eukaryotic

origin).

80S ribosome has 2 sites,

1. P-site contains the peptidyl-tRNA

2. A- site contains new amino acyl-tRNA.

Aminoacyl-tRNA for first codon: Met-tRNA enters at

P-site, leaving the A-site free and elongation cycle is

ready to start.

New aminoacyl-tRNA + GPT + EF-1 → complex that

enters site A, then EF-1 and GDP + Pi are released.

Peptidyl transferase catalyzes peptide bond

formation .

Uncharged tRNA moves from P to E site & then is

released.

The newly formed peptidyl- tRNA at the A-site

moves to P-site in the presence of GTP and EF-2

(translocase).

Now, the new aminoacyl-tRNA can come to A-site.

eEF-1 + GTP eEF-1 + GDP + Pi

It occurs when one of the chain terminating codons

(UAA, UAG or UGA) appears in the A-site.

No tRNA can recognize this codon, the releasing

factor + GTP+ peptidyl-tRNA cause hydrolysis of the

bond between the peptide and tRNA at the P-site.

80S ribosome is broken into 60S + 40S to finally

give separate peptide, tRNA, 40S, 60S, releasing

factor and GDP+Pi.

Polysomes (polyribosomes)

Multiple ribosomes on the same mRNA molecule

form a polyribosome, or «polysome» that can

translate the same mRNA molecule simultaneously.

Because of their relatively large size, the ribosome

particles cannot attach to an mRNA any closer than

35 nucleotides apart.

Post-translational Modification of

Polypeptide Chains

Many polypeptide chains are covalently modified,

either while they are still attached to the ribosome or

after their synthesis has been completed.

These modifications may include:

1. Removing part of the translated sequence.

2. Covalent addition of one or more chemical

groups required for protein activity.

Trimming

Many secretory proteins are initially made as large,

precursor molecules that are functionally inactive.

These precursor proteins are cleaved in the ER, the

Golgi apparatus, in developing secretory

vesicles (e.g. insulin), or after secretion.

e.g. Zymogens are inactive precursors of secreted

enzymes that become activated through cleavage

once they have reached their proper sites of action;

pepsinogen, becomes activated in the stomach.

The synthesis of enzymes as zymogens protects

the cell from being digested by its own products.

Covalent alterations

Proteins, both enzymatic and structural, may be

activated or inactivated by covalent attachment of a

variety of chemical groups:

1. Phosphorylation:

2. Glycosylation

3. hydroxylation

1. Phosphorylation:

It occurs on OH of serine,

threonine, or less frequently,

tyrosine residues in a

protein.

It is catalyzed by protein

kinases and may be

reversed by protein

phosphatases.

The phosphorylation may

increase or decrease the

functional activity of the

protein.

2. Glycosylation:

Proteins destined to become

part of a plasma membrane or

secreted from the cell have

carbohydrate chains attached

to serine or threonine OH (O-

linked) or asparagine (N-

linked).

The stepwise addition of sugars

occurs in the endoplasmic

reticulum and the Golgi

apparatus.

3. Hydroxylation

Proline and lysine of the α-chains of collagen are

extensively hydroxylated in the endoplasmic

reticulum.