INTRODUCTION.

The genetic message carried on the DNA molecule is a code. This code is ultimately
translated into a sequence of amino acids that, when complete, becomes a protein. Proteins
carry out the “business” of the cell. Some proteins are used as structural components of cells,
some are used to transport other molecules, still others are charged with directing chemical
reactions. The latter class of proteins is the enzymes. Regardless of the role played by a
protein in the cell one aspect is the same, they are all encoded in the base sequences of DNA.
The path from DNA sequence to protein sequence is an elegant but complex process that is
composed of two major steps. The first is transcription, in which DNA is converted into a
mature messenger RNA (mRNA), and the second is translation, in which the base sequence
of the mRNA is “read” and converted into an amino acid sequence.

1. INITIATION AND TRIGGER.

There are three distinct primary messenger pathways to alter transcription.
A) The primary messenger crosses the cell membrane and bind to a nuclear receptors,
which then interact with DNA to alter gene expression. (e.g in steroid or thyroid
hormones)
B) B) A second pathway is the activation of cytoplasmic protein kinases that then moves
to the nucleus to phosphorylate a latent transcription factor for activation.
C) A third common pathway is the activation of a latent transcription factor in the
cytosol, which then migrates to the nucleus and alters transcription.
In all cases, the binding of the activated transcription factor to DNA alters the transcription
of mRNAs encoded by the gene to which it binds.

2. TRANSCRIPTION.
 Transcription is the process in which a gene's DNA sequence is copied (transcribed)
to make an RNA molecule.
 RNA polymerase is the main transcription enzyme.
 Transcription begins when RNA polymerase binds to a promoter sequence near the
beginning of a gene (directly or through helper proteins).
 RNA polymerase uses one of the DNA strands (the template strand) as a template to
make a new, complementary RNA molecule.
 Transcription ends in a process called termination. Termination depends on sequences
in the RNA, which signal that the transcript is finished.
Transcription is the first step of gene expression. During this process, the DNA sequence of
a gene is copied into RNA.
Before transcription can take place, the DNA double helix must unwind near the gene that is
getting transcribed. The region of opened-up DNA is called a transcription bubble.

Transcription uses one of the two exposed DNA strands as a template; this strand is called
the template strand. The RNA product is complementary to the template strand and is
almost identical to the other DNA strand, called the nontemplate (or coding) strand.
However, there is one important difference: in the newly made RNA, all of the Thymine
nucleotides are replaced with Uracil nucleotides.

RNA polymerase.
RNA polymerases are enzymes that transcribe DNA into RNA. Using a DNA template,
RNA polymerase builds a new RNA molecule through base pairing. For instance, if there
is a G in the DNA template, RNA polymerase will add a C to the new, growing RNA
strand.
RNA polymerase always builds a new RNA strand in the 5’ to 3’ direction. That is, it can
only add RNA nucleotides (A, U, C, or G) to the 3' end of the strand. The complementary
strand is read in a 3’ to 5’.
Transcription initiation
To begin transcribing a gene, RNA polymerase binds to the DNA of the gene at a region
called the promoter region. This is called the TATA BOX. Basically, the promoter tells
the polymerase where to "sit down" on the DNA and begin transcribing.

Each gene has its own promoter. A promoter contains DNA sequences that let RNA
polymerase or its helper proteins attach to the DNA. Once the transcription bubble has
formed, the polymerase can start transcribing.
Transcription Elongation.
Once RNA polymerase is in position at the promoter, the next step of transcription—
elongation—can begin. Basically, elongation is the stage when the RNA strand gets
longer, thanks to the addition of new nucleotides.
During elongation, RNA polymerase "walks" along one strand of DNA, known as the
template strand, in the 3' to 5' direction. For each nucleotide in the template, RNA
polymerase adds a matching (complementary) RNA nucleotide to the 3' end of the RNA
strand. The RNA transcript is nearly identical to the non-template, or coding, strand of
DNA. However, RNA strands have the base uracil (U) in place of thymine (T), as well as
a slightly different sugar in the nucleotide. So, as we can see in the diagram below, each T
of the coding strand is replaced with a U in the RNA transcript.
Transcription termination
RNA polymerase will keep transcribing until it gets signals to stop. The process of ending
transcription is called termination, and it happens once the polymerase transcribes a
sequence of DNA known as a terminator. This happens when it encounters a new sequence
of DNA nucleotides called the chain-terminating sequence, which causes the polymerase
and the newly formed RNA chain to break away from the DNA strand.

POST-TRANSCRIPTIONAL MODIFICATION.
Post-transcriptional modification is a set of biological processes common to most eukaryotic
cells by which an RNA primary transcript is chemically altered following transcription from
a gene to produce a mature, functional RNA molecule that can then leave the nucleus and
perform any of a variety of different functions in the cell. There are many types of post-
transcriptional modifications achieved through a diverse class of molecular mechanisms.
Perhaps the most notable example is the conversion of precursor messenger RNA transcripts
into mature messenger RNA that is subsequently capable of being translated into protein.
This process includes three major steps that significantly modify the chemical structure of the
RNA molecule:
1. The addition of a 5' cap.
2. The addition of a 3' polyadenylated tail.
3. RNA splicing.
Such processing is vital for the correct translation of eukaryotic genomes because the initial
precursor mRNA produced by transcription often contains both exons (coding sequences) and
introns (non-coding sequences); splicing removes the introns and links the exons directly,
while the cap and tail facilitate the transport of the mRNA to a ribosome and protect it from
molecular degradation.

 5' processing.
Capping of the pre-mRNA involves the addition of 7-methylguanosine to the 5' end.
The cap protects the 5' end of the primary RNA transcript from attack by
ribonucleases.
 3' processing.
Cleavage and polyadenylation.
The pre-mRNA processing at the 3' end of the RNA molecule involves cleavage of its
3' end and then the addition of about 250 adenine residues to form a poly(A) tail. The
cleavage and adenylation reactions occur if a polyadenylation signal sequence (5'-
AAUAAA-3') is located near the 3' end of the pre-mRNA molecule, which is
followed by another sequence, which is usually (5'-CA-3') and is the site of cleavage.
PolyAdenylate polymerase then adds about 200 adenine units to the new 3' end of the
RNA molecule using ATP as a precursor. As the poly(A) tail is synthesised, it binds
multiple copies of poly(A) binding protein, which protects the 3'end from
ribonuclease digestion.
 Introns Splicing.
RNA splicing is the process by which introns, regions of RNA that do not code for
proteins, are removed from the pre-mRNA and the remaining exons connected to re-
form a single continuous molecule. Exons are sections of mRNA which become
"expressed" or translated into a protein. They are the coding portions of a mRNA
molecule. The splicing reaction is catalyzed by a large protein complex called the
spliceosome assembled from proteins and small nuclear RNA molecules that
recognize splice sites in the pre-mRNA sequence.
3. TRANSLATION.
Translation involves “decoding” a messenger RNA (mRNA) and using its information to
build a polypeptide, or chain of amino acids. For most purposes, a polypeptide is basically
just a protein (with the technical difference being that some large proteins are made up of
several polypeptide chains).
The genetic code
In an mRNA, the instructions for building a polypeptide come in groups of three nucleotides
called codons.
 There are 61 different codons for amino acids.
 Three “stop” codons mark the polypeptide as finished.
 One codon, AUG, is a “start” signal to kick off translation (it also specifies the amino
acid methionine)
These relationships between mRNA codons and amino acids are known as the genetic code.
Codons to amino acids.
In translation, the codons of an mRNA are read in order (from the 5' end to the 3' end) by
molecules called transfer RNAs, or tRNAs.
Each tRNA has an anticodon, a set of three nucleotides that binds to a matching mRNA
codon through base pairing. The other end of the tRNA carries the amino acid that's specified
by the codon.

tRNAs bind to mRNAs inside of a protein-and-RNA structure called the ribosome. As

tRNAs enter slots in the ribosome and bind to codons, their amino acids are linked to the
growing polypeptide chain in a chemical reaction. The end result is a polypeptide whose
amino acid sequence mirrors the sequence of codons in the mRNA.
TRANSLATION: INITIATION
In order for translation to start, we need a few key ingredients. These include:
 A ribosome (which comes in two pieces, large 60S and small 40S)
 An mRNA with instructions for the protein we'll build
 An "initiator" tRNA carrying the first amino acid in the protein, which is almost
always methionine (Met)
During initiation, these pieces must come together in just the right way. Together, they form
the initiation complex, the molecular setup needed to start making a new protein. The events
translation initiation are the binding of the small ribosomal subunit to the mRNA molecule
and the binding of the charged tRNA that bears the first amino acid of the polypeptide chain.
The 5´ methyl cap of the mRNA is involved in binding the small ribosomal subunit. The
small ribosomal subunit, mRNA and tRNA complex recruits the large ribosomal subunit.
• The large subunit of the ribosome contains three binding sites
Amino acyl (A site)
Peptidyl (P site)
Exit (E site)
• At initiation,
The tRNA occupies the P site.
A second, charged tRNA complementary to the next codon binds the A site.
TRANSLATION: ELONGATION.
Methionine-carrying tRNA starts out in the middle slot of the ribosome, called the P site.
Next to it, a fresh codon is exposed in another slot, called the A site. The A site will be the
"landing site" for the next tRNA, one whose anticodon is a perfect (complementary) match
for the exposed codon.
Once the matching tRNA has landed in the A site, there is the formation of the peptide bond
that connects one amino acid to another. This step transfers the methionine from the first
tRNA onto the amino acid of the second tRNA in the A site. The methionine forms the N-
terminus of the polypeptide, and the other amino acid is the C-terminus. Elongation can be
thought to involve three processes: 1) aligning each aminoacylated tRNA, 2) forming the
peptide bond to add the new amino acid to the polypeptide chain, and 3) moving the
ribosome along the mRNA by three more bases (one codon). EF-1alpha aligns each new
tRNA into the A site of the large subunit. This process requires the hydrolysis of GTP. When
the A site is filled, a peptidyl transferase activity catalyzes the formation of the peptide bond
between the amino acid in the A site and the adjacent amino acid in the P site. The formation
of the peptide bond requires several proteins and RNA molecules of the large subunit and
some evidence suggests that the peptide bond formation is catalyzed by an RNA molecule.
After the peptide bond is formed the ribosome translocates 3 bases, the tRNA from the P-site
that is no longer attached to its amino acid moves to the E site, and the tRNA with amino acid
from the A site is now occupies the P site. The A site is now empty. This is known as the
“posttranslocation state” for the ribosome.

TRANSLATION: TERMINATION.
Elongation proceeds until a stop codon is reached. There are three stop codons in the genetic
code: UAG, UGA, UAA. There is no tRNA that bears an anticodon that can read any of the
stop codons (the anticodons would be AUC, ACU, or AUU). Therefore, no tRNA can fill the
empty A site of the elongation complex. Instead the A site is filled by a release factor, and the
amino acid is cleaved from the tRNA that occupies the P site of the ribosome.

POST-TRANSLATIONAL MODIFICATION.
Protein post-translational modifications (PTMs) increases the functional diversity of the
protein by the covalent addition of functional groups or proteins, proteolytic cleavage of
regulatory subunits, or degradation of entire proteins. These modifications include
phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation,
lipidation and proteolysis.
1. Phosphorylation.
Protein phosphorylation is the addition of a phosphate group principally on
serine, threonine or tyrosine residues. Phosphorylation plays critical roles in the
regulation of many cellular processes, including cell cycle, growth, apoptosis and
signal transduction pathways.
2. Glycosylation.
Is the addition of a glycosyl group to either arginine, asparagine, cysteine,
hydroxylysine, serine, threonine, tyrosine, or tryptophan resulting in a
glycoprotein. Distinct from glycation, which is regarded as a nonenzymatic
attachment of sugars. It has significant effects on protein folding, conformation,
distribution, stability and activity.
 3. Ubiquitination.
the covalent linkage to the protein ubiquitin.
4. Methylation.
The transfer of one-carbon methyl groups to nitrogen or oxygen (N- and O-
methylation, respectively) to amino acid side chains increases the hydrophobicity
of the protein and can neutralize a negative amino acid charge when bound to
carboxylic acids. Methylation is mediated by methyltransferases, and S-adenosyl
methionine (SAM) is the primary methyl group donor.
5. N-acetylation.
The addition of an acetyl group, either at the N-terminus of the protein or at
lysine residues.
6. Lipidation.
Lipidation is a method to target proteins to membranes in organelles
(endoplasmic reticulum [ER], Golgi apparatus, mitochondria), vesicles
(endosomes, lysosomes) and the plasma membrane. The four types of lipidation
are:
 C-terminal glycosyl phosphatidylinositol (GPI) anchor
 N-terminal myristoylation
 S-myristoylation
 S-prenylation
Each type of modification gives proteins distinct membrane affinities, although
all types of lipidation increase the hydrophobicity of a protein and thus its affinity
for membranes. The different types of lipidation are also not mutually exclusive,
in that two or more lipids can be attached to a given protein.
7. Proteolysis.
Peptide bonds are indefinitely stable under physiological conditions, and
therefore cells require some mechanism to break these bonds. Proteases comprise
a family of enzymes that cleave the peptide bonds of proteins and are critical in
antigen processing, apoptosis, surface protein shedding and cell signaling.
8. Hydroxylation.
Is the addition of an oxygen atom to the side-chain of a Pro or Lys residue.

4. DETERMINATION AND IDENTIFICATION OF

PROTEIN DESTINATION.
Different proteins need to be sent to different parts of a cell, or, in some cases, exported out
of the cell and into the extracellular space. Cells have various shipping systems, kind of like
molecular versions of the postal service, to make sure that proteins arrive at their correct
destinations. In these systems, molecular labels (often, amino acid sequences) are used to
"address" proteins for delivery to specific locations.

CELLULAR SHIPPING ROUTES.

Translation of all proteins in a cell begins in the cytosol (except for a few proteins made in
mitochondria and chloroplasts). As a protein is made, it passes step by step through a
shipping "decision tree." At each stage, the protein is checked for molecular tags to see if it
needs to be re-routed to a different pathway or destination.

The first major branch point comes shortly after translation starts. At this point, the protein
will either remain in the cytosol for the rest of translation or be fed into the endoplasmic
reticulum (ER) as it is translated.
 Proteins are fed into the ER during translation if they have an amino sequence called a
signal peptide. In general, proteins bound for organelles in the endomembrane system
(such as the ER, Golgi apparatus, and lysosome) or for the exterior of the cell must
enter the ER at this stage.
 Proteins that do not have a signal peptide stay in the cytosol for the rest of translation.
If they lack other "address labels," they'll stay in the cytosol permanently. However, if
they have the right labels, they can be sent to the mitochondria, chloroplasts,
peroxisomes, or nucleus after translation.
Transport through the endomembrane system.
In the ER, proteins fold into their correct shapes, and may also get sugar groups attached to
them. Most proteins are then transported to the Golgi apparatus in membrane vesicles. Some
proteins, however, need to stay in the ER and do their jobs there. These proteins have amino
acid tags that ensure they are shipped back to the ER if they "escape" into the Golgi.
In the Golgi apparatus, proteins may undergo more modifications (such as addition of sugar
groups) and before going on to their final destinations. These destinations include lysosomes,
the plasma membrane, and the cell exterior. Some proteins need to do their jobs in the Golgi
(are "Golgi-resident), and a variety of molecular signals, including amino acid tags and
structural features, are used to keep them there or bring them back.
If they don't have any specific tags, proteins are sent from the Golgi to the cell surface, where
they’re secreted to the cell exterior (if they’re free-floating) or delivered to the plasma
membrane (if they’re membrane-embedded).
Proteins are shipped to other destinations if they contain the right molecular labels. For
example, proteins destined for the lysosome have a molecular tag consisting of a sugar with a
phosphate group attached. In the Golgi apparatus, proteins with this tag are sorted into
vesicles bound for the lysosome.
Targeting to non-endomembrane organelles.
Proteins that are made in the cytosol (don't enter ER during translation) may stay
permanently in the cytosol. However, they may also be shipped to other, non-endomembrane
destinations in the cell. For instance, proteins bound for the mitochondria, chloroplasts,
peroxisomes, and nucleus are usually made in the cytosol and delivered after translation is
complete.
To be delivered to one of these organelles after translation, a protein must contain a specific
amino acid "address label." The label is recognized by other proteins in the cell, which help
transport the protein to the right destination. For instance, Proteins needed in the peroxisome
have a specific sequence of amino acids called a peroxisomal targeting signal. The classic
signal consists of just three amino acids, serine-lysine-leucine, found at the very end (C-
terminus) of a protein. This pattern of amino acids is recognized by a helper protein in the
cytosol, which brings the protein to the peroxisome.