Coding nucleotide sequences of DNA and mRNA and the amino acid

sequence of a polypeptide chain

 Chromosomes
 Nearly every body cell contains, within its nucleus, an identical copy of the
entire complement of the individual’s genetic material. Two important
exceptions are red blood cells (which have no nucleus) and the gametes or
sex cells.
 In a resting cell, the chromatin ( is diffuse and hard to see under the
microscope, but when the cell prepares to divide, it is collected into highly
visible, compact, sausage-shaped structures called chromosomes.
 Each chromosome is one of a pair, one inherited from the mother and one
from the father, so the human cell has 46 chromosomes that can be
arranged as 23 pairs.
 A cell with 23 pairs of chromosomes is termed diploid. Gametes
(spermatozoa and ova) with only half of the normal complement, i.e. 23
chromosomes instead of 46, are described as haploid.

 Chromosomes belonging to the same pair are called homologous

chromosomes
  Each pair of chromosomes is numbered, the largest
pair being no. 1.
 The first 22 pairs are collectively known as autosomes,
and the chromosomes of each pair are identical.
 The chromosomes of pair 23 are called the sex
chromosomes because they determine the individual’s
gender

 Unlike autosomes, these two chromosomes are not

necessarily identical; the Y chromosome is much shorter
than the X and is carried only by males. A child inheriting
two X chromosomes (XX), one from each parent, is
female, and a child inheriting an X from his mother and a
Y from his father (XY) is male
 Definition of genes

A specific sequence of nucleotides in DNA or RNA that is

located usually on a chromosome and that is the functional
unit of inheritance controlling the transmission and
expression of one or more traits by specifying the structure
of a particular polypeptide and especially a protein or
controlling the function of other genetic material.
 Along the length of the chromosomes are the genes.

 Each gene contains information in code that allows the cell to make (almost always)
a specific protein, the so-called gene product.

 Each gene codes for one specific protein, and research puts the number of genes
within the human genome at 24 500 that means 24 500 different proteins.

 Genes normally exist in pairs, because each gene site (locus) is present at
corresponding sites on both homologous chromosomes.
Nucleotides consist of three subunits:

 sugar
 phosphate group
 base.

 The DNA molecule is sometimes likened to a twisted ladder, with the

uprights formed by alternating chains of sugar and phosphate units .

 In DNA, the sugar is deoxyribose, thus DNA. The bases are linked to
the sugars, and each base binds to another base on the other
sugar/phosphate chain, forming the rungs of the ladder.

 The two chains are twisted around one another, giving a double helix
(twisted ladder) arrangement. The double helix itself is further twisted
and wrapped in a highly organised way around structural proteins
called histones, which are important in maintaining the heavily coiled
three-dimensional shape of the DNA
 Each base along one strand of DNA
pairs with a base on the other strand in
a precise and predictable way. This is
known as complementary base pairing.
Adenine always pairs with thymine (and
vice versa), and cytosine and guanine
always go together.
 STRUCTURE OF DNA

 The function of DNA is carrying huge amounts of information that

determines all biological activities of an organism, and which is
transmitted from one generation to the next. The key to how this
information is kept is found in the bases within DNA. There are four
bases:

 Adenine (A)
 Guanine (G)
 Thymine (T)
 Cytosine (C).

They are arranged in a precise order along the DNA molecule, making
a code that can be read when protein synthesis is required.
 GENETIC CODE

 A four-letter language is translated to 20 letter language during protein

synthesis. There should be a specific relationship among the four bases
of DNA and sequence of 20 amino acids in the protein.
 Genetic code is the term that used for the way that the four bases of
DNA--the A, C, G, and Ts--are strung together in a way that the cellular
machinery, the ribosome, can read them and turn them into a protein.

 In the genetic code, each three nucleotides in a row count as a triplet

and code for a single amino acid.

 So each sequence of three codes for an amino acid. And proteins are
made up of sometimes hundreds of amino acids. So the code that would
make one protein could have hundreds, sometimes even thousands, of
triplets contained in it.
GENETIC CODE TABLE
 Properties of Genetic Code

•Triplet code

•Non-ambiguous and Universal

•Degenerate code

•Nonoverlapping code

•Commaless

•Start and Stop Codons

•Polarity
Triplet code
A codon or a code word is defined as a group of bases that
specify an amino acid. There is strong evidence, which
proves that a sequence of three nucleotides codes for an
amino acid in the protein, i.e., the code is a triplet.
The four bases of nucleotide i.e, (A, G, C, and U) are used
to produce three-base codons. The 64 codons involve sense
codons (that specify amino acids). Hence, there are 64
codons for 20 amino acids since every codon for one amino
acid means that there exist more than code for the same
amino acid.
Commaless code
No room for punctuation in between which indicates that
every codon is adjacent to the previous one without any
nucleotides between them.
.
Nonoverlapping code
The code is read sequentially in a group of three and a nucleotide which becomes a
part of triplet never becomes part of the next triplet.
For example
5’-UCU-3’ codes for Serine
5’-AUG-3’ codes for methionine
Polarity
Each triplet is read from 5’ → 3’ direction and the beginning base is 5’ followed by the
base in the middle then the last base which is 3’. This implies that the codons have
a fixed polarity and if the codon is read in the reverse direction, the base sequence of
the codon would reverse and would specify two different proteins.

Degenerate code
Every amino acid except tryptophan (UGG) and methionine (AUG) is coded by various
codons, i.e, a few codons are synonyms and this aspect is known as the degeneracy
of genetic code. For instance, UGA codes for tryptophan in yeast mitochondria.
Start and Stop Codons:
Generally, AUG codon is the initiating or start codon. The polypeptide chain
starts either with eukaryotes (methionine) or prokaryotes (N-
formylmethionine).
On the other hand, UAG, UAA and UGA are called as termination codons or
stop codons. These are not read by any tRNA molecules and they never
code for any amino acids.

Non-ambiguous and Universal:

The genetic code is non-ambiguous which means a specific codon will only
code for a particular amino acid. Also, the same genetic code is seen valid
for all the organisms i.e. they are universal
 Regulation of gene expression

 Eukaryotic genes are not organized into operons, so each

gene must be regulated independently.

 In addition, eukaryotic cells have many more genes than

prokaryotic cells.

 Regulation of gene expression can happen at any of the

stages as DNA is transcribed into mRNA and mRNA is
translated into protein.

 For convenience, regulation is divided into five levels:

epigenetic, transcriptional, post-transcriptional, translational,
and post-translational.
 Epigenetic control involves changes to genes that do not alter the nucleotide
sequence of the DNA and are not permanent.

 Instead, these changes alter the chromosomal structure so that genes can be turned
on or off. This level of control occurs through heritable chemical modifications of the
DNA and/or chromosomal proteins.

 One example of chemical modifications of DNA is the addition of methyl groups to the
DNA, in a process called methylation.

 In general, methylation suppresses transcription.

 If a gene is to be transcribed, the nucleosomes
surrounding that region of DNA can slide down
the DNA to open that specific chromosomal
region and allow access for RNA polymerase
and other proteins, called transcription factors,
to bind to the promoter region and initiate
transcription.

 If a gene is to remain turned off, or silenced, the

histone proteins and DNA have different
modifications that signal a closed chromosomal
configuration
 Transcription
Transcription is the first part of the central dogma of molecular biology: DNA
→ RNA. It is the transfer of genetic instructions in DNA to mRNA.

 Transcription happens in the nucleus of the cell.

 During transcription, a strand of mRNA is made that is complementary to a

strand of DNA called gene.

 A gene contains the basic three regions, promoter, coding sequence,

and terminator
Major components of gene
 Like prokaryotic cells, the transcription of genes in eukaryotes
requires an RNA polymerase to bind to a promoter to initiate
transcription.

 In eukaryotes, RNA polymerase requires other proteins,

or transcription factors, to facilitate transcription initiation.

 Transcription factors are proteins that bind to the promoter

sequence and other regulatory sequences to control the
transcription of the target gene.

 RNA polymerase by itself cannot initiate transcription in eukaryotic

cells.

 Transcription factors must bind to the promoter region first and

recruit RNA polymerase to the site for transcription to begin.
 The Promoter and Transcription Factors

 In eukaryotic genes, the promoter region is immediately upstream of the

coding sequence.

 This region can range from a few to hundreds of nucleotides long. The length
of the promoter is gene-specific and can differ dramatically between genes.

 The longer the promoter, the more available space for proteins to bind.

 The purpose of the promoter is to bind transcription factors that control the
initiation of transcription.
 The Promoter and Transcription Factors

 Within the promoter region, just upstream of the transcriptional start

site, resides the TATA box.

 This box is simply a repeat of thymine and adenine dinucleotides

(literally, TATA repeats).

 Transcription factors bind to the TATA box, assembling an initiation

complex. Once this complex is assembled, RNA polymerase binds to
its upstream sequence and becomes phosphorylated.
 Enhancers and Repressors
 In some eukaryotic genes, there are regions that help increase
transcription.

 These regions, called enhancers, are not necessarily close to the genes;
they can be located thousands of nucleotides away.

 They can be found upstream, within the coding region, or downstream of a

gene.

 Enhancers are binding sites for activators.

 When an enhancer is far away from a gene, the DNA folds such that the
enhancer is brought into proximity with the promoter, allowing interaction
between the activators and the transcription initiation complex
 Post-transcriptional Control of Gene Expression
 Post-transcriptional regulation occurs after the mRNA is transcribed but
before translation begins.

 This regulation can occur at the level of mRNA processing, transport from
the nucleus to the cytoplasm, or binding to ribosomes.

 Alternative RNA splicing is a mechanism that allows different combinations

of introns, and sometimes exons, to be removed from the primary
transcript.
 This allows different protein products to be produced from one gene.
 Alternative splicing can act as a mechanism of gene regulation.
Differential splicing is used to produce different protein products in different
cells or at different times within the same cell.
Splicing removes introns from mRNA
 siRNA (Small Interfering RNA)

The siRNA or small interfering RNA is a 22 to 25 base pair long smaller

molecules of dsRNA having a dinucleotide overhang at the 3’ end,
interfere in the protein synthesis by blocking the translation
 The siRNA is also known as small interfering ribonucleic
acid or silencing RNA and is a molecule that prevents gene
expression.

 The entire process of gene silencing through the siRNA is called

a mechanism of RNA interference or siRNA knockdown.
 Structure of siRNA
 The siRNA is double-stranded, short
and 20 to 25 nucleotides long.

 One of the unique characters of the

siRNA is the presence of the 3’ OH
dinucleotide overhang.

 From the double-strand, one strand is

known as a guided strand while the
other is known as a passenger strand.

 It is also called a sense strand and

antisense strand, respectively.
 Mechanism of siRNA action
 Functionally, the siRNA degrades the growing
mRNA (exogenous as well as endogenous) and
stops gene expression.

 Once the siRNA inserts into our cell, the

specialized protein called ‘dicer’ having tetrameric
manganese ions cuts or cleaves the dsRNA into
smaller pieces.

 These smaller fragments of dsRNA then

incorporated into the protein complex having
multiple subunits and form the RNAi-induced
silencing complex, RISC.

 The RISC finds the appropriate mRNA target and

cleaved it by a combination of endo and
exonuclease activity
 Applications of siRNA

 siRNA-mediated gene silencing method to suppress cancer-causing

genes.

 It is also employed in pathway analysis and pathway identifications

like cytokinesis, insulin signaling and cell defense mechanism, etc.
 micro RNA(miRNA)

 miRNA and siRNA are biochemically and functionally

indistinguishable.

 Both are 19-20 nucleotides (nt) in length with 5’-phosphate and 3’-
hydroxyl ends, and assemble into RISC to silence specific gene
expression .

 These molecules are distinguished based on their respective

origins.

 MicroRNA is derived from the double-stranded region of a 60-70nt

RNA hairpin precursor while siRNA is generated from long double-
stranded RNA (dsRNA).
 MiRNA are small, evolutionary conserved,
single-stranded, non-coding RNA molecules
that bind target mRNA to prevent protein
production.

 Mature miRNA is generated through two-

step cleavage of primary miRNA (pri-
miRNA), which incorporates into the effector
complex RNA-induced silencing complex
(RISC).
 The miRNA functions as a guide by
base-pairing with target mRNA to
negatively regulate its expression.

 The level of complementarity between

the guide and mRNA target determines
which silencing mechanism will be
employed.

 Cleavage of target messenger RNA

(mRNA) with subsequent degradation or
translation inhibition
 Cell cycle and its regulation

 Some cells such as neurons and skeletal muscle cells spend all their
lifetime in G0 whereas others, including bone marrow cells and the
epithelium of the gastrointestinal tract, divide daily.

 cell cycle is an ordered sequential series of phases:

•G1: preparation for DNA synthesis

•S: DNA synthesis and chromosome duplication

• G2: preparation for division

•M: mitosis, division into two daughter cells

 Cell division requires the controlled timing of the critical S phase and M phases.

 Entry into each of these phases is tightly regulated at check points (restriction points) at the start of
the S and M phases.

 The impetus for a cell to enter the cycle ( move from G0 into G1) may be growth factors acting on
growth factor receptors.

 Growth factor stimulate the synthesis of both positive and negative regulator that control the
changes necessary for cell division.
 POSITIVE REGULATORS OF THE CELL CYCLE

 The cycle begins when a growth factor acts on a quiescent cell,

provoking it to divide.

 Growth factors stimulate production of two families of proteins,

namely cyclins and serine/threonine protein kinases called cyclin-
dependent kinases (cdks).

 The cdks sequentially phosphorylate various enzymes – activating

some and inhibiting others – to coordinate the progression of the cell
through the cycle.

 Cyclin A activates cdks 1 and 2; cyclin B, cdk 1; cyclin D, cdks 4and 6;

and cyclin E, cdk 2.
 The activity of these cyclin/cdk complexes is negatively modulated at one or other of the two check
points.

 In quiescent G0 cells, cyclin D is present in low concentration, and an important regulatory protein –
the Rb protein is hypophosphorylated.

 This restrains the cell cycle at check point 1 by inhibiting the expression of several proteins critical
for further cycle progression.

 Growth factor action on a cell in G0 propels it into G1, which prepares the cell for S phase.

 The concentration of cyclin D increases and the cyclin D/cdk complex phosphorylates and activates
the proteins required for DNA replication
 NEGATIVE REGULATORS OF THE CELL CYCLE

 One of the main negative regulators is the Rb protein, which restrains the cell cycle while it is
hypophosphorylated.

 Inhibitors of the cdks also serve as negative regulators, their main action being at check point 1.

 There are two known families of inhibitors: the CIP family (cdk inhibitory proteins)– proteins p21, p27 and p57;
and the Ink family (inhibitors of kinases) – proteins p16, p19 and p15.

 Protein p21 is under the control of the p53 gene – a particularly important negative regulator which is relevant
in carcinogenesis – that operates at check point 1.
 Inhibition of the cycle at check point 1

 The p53 gene has been called the ‘guardian of the genome’.

 It codes for the p53 protein, a transcription factor found in only low concentrations in normal healthy
cells.

 However, following DNA damage, the protein accumulates and activates the transcription of several
genes, one of which codes for p21.

 Protein p21 inactivates cyclin/cdk complexes, thus preventing Rb phosphorylation, which means that
the cycle is arrested at check point 1.

 This allows for DNA repair. If the repair is successful, the cycle proceeds past check point 1 into S phase.
 Apoptosis

 Apoptosis is programmed cell death. It is an essential biological process and critical for (e.g.)
embryogenesis and tissue homeostasis.

 Apoptosis depends upon a cascade of proteases called caspases.

 It is regulated by a built-in genetically programmed self-destruct mechanism consisting of sequence of

biochemical events.

 Apoptosis plays an essential role in embryogenesis, shaping organs during development by eliminating
cells that have become redundant.

 Impaired apoptosis is also implicated in the pathophysiology of many condition, chronic

neurodegenerative diseases such as Alzheimer’s, multiple sclerosis and Parkinson’s disease
 Stimulation of death receptors by external
ligands (the extrinsic pathway) and an
internal mitochondrial pathway.

 Both routes activate initiator caspases and

converge on a final common effector caspase
pathway.