How in a dihybrid cross independent assortment of genes leads to 50 % recombinant

gametes ? How can this recombinant percentage be less than 50 % ?

Genetic linkage is a term which describes the tendency of certain loci or alleles to be
inherited together. Genetic loci on the same chromosome are physically close to one another
and tend to stay together during meiosis, and are thus genetically linked.

During meiosis, chromosomes assort randomly into gametes, such that the segregation of
alleles of one gene is independent of alleles of another gene. This is stated in Mendel's
Second Law and is known as the law of independent assortment. The law of independent
assortment always holds true for genes that are located on different chromosomes, but for
genes that are on the same chromosome, it does not always hold true.

As an example of independent assortment, consider the crossing of the pure-bred homozygote

parental strain with genotype AABB with a different pure-bred strain with genotype aabb. A
and a and B and b represent the alleles of genes A and B. Crossing these homozygous
parental strains will result in F1 generation offspring with genotype AaBb. The F1 offspring
AaBb produces gametes that are AB, Ab, aB, and ab with equal frequencies (25%) because
the alleles of gene A assort independently of the alleles for gene B during meiosis. Note that
2 of the 4 gametes (50 %)—Ab and aB—were not present in the parental generation. These
gametes represent recombinant gametes. Recombinant gametes are those gametes that differ
from both of the haploid gametes that made up the diploid cell. In this example, the
recombination frequency is 50% since 2 of the 4 gametes were recombinant gametes.

The recombination frequency will be 50% when two genes are located on different
chromosomes or when they are widely separated on the same chromosome. This is a
consequence of independent assortment.

When two genes are close together on the same chromosome, they do not assort
independently and are said to be linked. Whereas genes located on different chromosomes
assort independently and have a recombination frequency of 50%, linked genes have a
recombination frequency that is less than 50%.

As an example of linkage, consider the classic experiment by William Bateson and Reginald
Punnett. They were interested in trait inheritance in the sweet pea and were studying two
genes—the gene for flower colour (P, purple, and p, red) and the gene affecting the shape of
pollen grains (L, long, and l, round). They crossed the pure lines PPLL and ppll and then self-
crossed the resulting PpLl lines. According to Mendelian genetics, the expected phenotypes
would occur in a 9:3:3:1 ratio of PL:Pl:pL:pl. To their surprise, they observed an increased
frequency of PL and pl and a decreased frequency of Pl and pL (see table below).

Their experiment revealed linkage between the P and L alleles and the p and l alleles. The
frequency of P occurring together with L and with p occurring together with l is greater than
that of the recombinant Pl and pL. The recombination frequency cannot be computed directly
from this experiment, but intuitively it is less than 50%.

Early experimental evidence suggesting that DNA is likely to be a duplex structure

Chemical analysis of DNA revealed that in DNA preparation obtained from different
organisms the quantity of adenine was equal to thymine and that of guanine with cytosine –
that is , A=T and G = C or A/T = 1 ,G/C = 1 . In contrast A+T/G+C ratio varied widely in
DNAs of different species. In bacteriophage T2 the ratio is 1.08 , in Micrococcus species 0.39
and human 1.53. This relationship between molar proportion of the bases was noted by Erwin
Chargaff and is known as Chargaff’s rule. The chemical structure of purine and pyrimidine
nucleotides furthermore suggested that a purine could associate with a pyrimidine through
hydrogen bonds .Hydrogen bonds are weak forces of attraction that are formed when a
positively charged H atom which is itself covalently bound to oxygen or nitrogen atom ,is
simultaneously attracted by a more negatively charged oxygen or nitrogen atom also
covalently bound to the adjoining molecule. It appeared that two H bonds could satisfy the
bonding affinity between A and T ,while pairing of G with C required three H bonds. This
together with the molar equivalence of A and T on one hand and that of G and C on the other
,provided two key leads to DNA model building – it was reasoned that perhaps DNA
molecule was a two stranded structure with the two strands held together by specific bonding
between the nitrogenous bases.

Data from X-ray diffraction studies suggested that the two strands were spirally twisted
around one another to form a helical structure.
Polarity of a nucleotide strand :

A polynucleotide has a 5’ end (top in the figure) and a 3’ end (bottom). The 5’ end carries
the free phosphate group and the 3’ end the hydroxyl group. Thus ,structurally the nucleotide
is different at its two ends and hence is said to be of opposite polarity. This property of the
single stranded DNA molecule proved to be of great significance in elucidation of the
structure of native DNA molecule.
Griffith's experiment

Griffith's experiment, conducted in 1928 by Frederick Griffith, was one of the first
experiments suggesting that bacteria are capable of transferring genetic information through a
process known as transformation.

Griffith used two strains of Diplococcus pneumoniae the bacteria that cause pneumonia , a
type III-S (smooth) and type II-R (rough) strain. The III-S strain covers itself with a
polysaccharide capsule that protects it from the host's immune system, resulting in the death
of the host and hence is virulent ,while the II-R strain doesn't have that protective capsule
and is defeated by the host's immune system and is thus avirulent.

The capsular characteristics differ in different strains of Diplococcus pneumoniae , but it is a

stable genetic trait in a particular strain and thus serves as a distinctive marker to classify the
bacteria in categories such as type II or type III etc. The difference between these types
resides in difference in chemical composition of the polysaccharide.

The experiment ,which was carried out in mice ,was briefly as follows :

 Living type II R avirulent bacteria were injected into mice . The mice as expected
suffered no illness and were alive
 Living type IIIS virulent bacteria injected similarly killed the mice and live bacteria
were recovered from blood.
 Killed type IIIS bacteria when injected produced no illness as expected and no live
bacteria could be recovered.
 A mixture of living type IIR and killed type IIIS when injected killed the mice and
interestingly living type IIIS bacteria were recovered when in fact killed Type IIIS
were injected.

How living Type IIIS bacteria were produced in the dead mice ?????

Griffith concluded that the type II-R had been "transformed" into the lethal type III-S strain
by a " principle" that was somehow part of the dead type III-S strain bacteria. The underlying
principle was thus referred to as ‘transforming principle’
Proof that the ‘Transforming Principle’ is DNA

The first evidence showing that the transforming principle was DNA was published by O.T.
Avery ,C.M .Macleod and M .McCarty in 1944. They showed that if highly purified DNA
from Type IIIS pneumococci was present with Type IIR pneumococci , some of the
pneumococci were transformed to Type IIIS. But how could one be sure that the DNA was
really pure ? May be the DNA preparation contained a few molecules of protein and these
contaminating proteins were responsible for the observed transformation. The most definitive
experiments in Avery ,MacLeod and McCarty’s proof that DNA was the transforming
principle involved the use of enzymes that degrade DNA ,RNA or protein. In separate
experiments ,highly purified DNA from Type IIIS cells was treated with (1)
deoxyribonuclease ( DNase , which degrades DNA ) ,(2) ribonuclease (RNase ,which
degrades RNA ) or (3) proteases (which degrade proteins) and then tested for its ability to
transform Type IIR cells to Type IIIS. Only DNase had any effect on the transforming
activity of the DNA preparation ; it totally eliminated all transforming activity. This
experiment strongly suggested that the chemical that corresponds to the transforming
principle was DNA

Hershey-Chase Experiment

The Hershey–Chase experiments were a series of experiments conducted in 1952 by Alfred

Hershey and Martha Chase, confirming that DNA was the genetic material, which had first
been demonstrated in the 1944 Avery–MacLeod–McCarty experiment. While DNA had been
known to biologists since 1869, most assumed at the time that proteins carried the
information for inheritance.

Hershey and Chase conducted their experiments on the T2 phage , a virus that infects and
multiplies in the bacterium E coli .Bacteriophage T 2 contains a hexagonal head of made up of
protein in which the viral nucleic acid DNA is present.

The basis for the Hershey-Chase experiment is that DNA contains phosphorus but no
sulfur ,whereas proteins contain sulfur but no phosphorus. Thus , Hershey and Chase were
able to specifically label either (1) the phage DNA by growth in a medium containing the
radioactive isotope of phosphorus ,32P ,in place of the normal isotope , 31P or (2) the phage
protein coats by growth in a medium containing radioactive sulfur , 35S ,in place of the normal
isotope ,32S.

When T2 phage particles labeled with 35S were mixed with E coli cells for a few minutes
and were then subjected to shearing forces by placing the infected cells in a high speed
blender , it was found that most of the radioactivity found only outside of the cell but with 32P
labeled phages , all the radioactivity was found inside the cells suggesting that it is only the
DNA of virus not protein which enters the bacteria where it directs the production of progeny
virus particles.
Replication fork :

The replication fork is a structure that forms within the nucleus during DNA replication. It is
created by helicases, which break the hydrogen bonds holding the two DNA strands together.
The resulting structure has two branching "prongs", each one made up of a single strand of
DNA. These two strands serve as the template for the leading and lagging strands which will
be created as DNA polymerase matches complementary nucleotides to the templates; The
templates may be properly referred to as the leading strand template and the lagging strand
template

Components of replication fork and their functions are summarized in table :

Components Functions
DNA helicase Unwinds DNA by catalyzing the breakdown of hydrogen bonds
DNA polymerase DNA synthesis ,repair of gaps in lagging strand
Single stranded binding protein Stabilizes single-stranded regions in replication fork
RNA primase Primes Okazaki fragment
DNA ligase Joins Okazaki fragments in lagging strand
RNase H Removes RNA primer from lagging strand
DNA topoisomerase Prevents supercoiling

Fig : Replication fork

lac operon
The lac operon is an operon required for the transport and metabolism of lactose in
Escherichia coli and some other enteric bacteria.

It consists of regulatory components and structural genes ,both comprising a stretch of

DNA lying close together. Three important regulatory components have been identified.
These are the regulatory gene designated as lacI and two smaller DNA sequences called
promoter p and the operator O. The three structural genes are: lacZ, lacY, and lacA.

lacZ encodes β-galactosidase, an intracellular enzyme that cleaves the disaccharide lactose
into glucose and galactose.

lacY encodes β-galactoside permease , a membrane-bound transport protein that pumps

lactose into the cell.

lacA encodes β-galactoside transacetylase, an enzyme that transfers an acetyl group from
acetyl-CoA to β-galactosides.

Synthesis of all 3 enzymes encoded in the lac operon is rapidly induced when E coli cells are
placed in a medium containing lactose as the only carbon source and repressed when the cells
are switched to a medium without lactose. Thus all three genes of lac operon are coordinately
regulated.

Fig : The lac operon showing the relative position of the regulatory components
and the structural genes

The regulatory gene i that codes for an mRNA is translated to produce a protein referred to as
the lac repressor. In the absence of lactose, the lac repressor binds to the operator . The
repressor binding to the operator prevents RNA polymerase from moving beyond the
promoter and therefore the genes for lactose catabolism are not transcribed into mRNA.
When lactose is present in the cell ,a small amount of it is converted to allolactose.
Allolactose binds to the lac repressor .This causes a conformational change in the lac
repressor. Thus altered, the repressor is unable to bind to the operator, allowing RNAP to
transcribe the lac genes, resulting in the production of the enzymes needed to metabolize
lactose. Since allolactose induces or stimulates transcription of the lac operon , it is called an
inducer.

This control prevents the cell from producing the enzymes required for lactose catabolism
because it would be inefficient to produce enzymes when lactose is not available or if there is
a more readily-available energy source such as glucose.

Transcription

DNA region at transcription initiation site :

Transcription is initiated at the control region of the gene which lies upstream from the
structural gene or genes (if these occur as an operon ). Typically, the control region consists
of a relatively long promoter , about 80 nucleotides (nt) long and an operator which is 27 nt
long. The two regions are contiguous and carry an overlapping common sequence of 8
nucleotides. Initiation of transcription occurs at a base which is an adenosine residue lying
within the promoter and whose position is denoted as position 0 with nucleotides lying
upstream denoted by a minus (-) sign. Two regions within the promoter are important in
transcription. One is the -10 region , also called the Pribnow box after its discoverer , which
stretches from position -6 to -12. In bacteria the Pribnow sequence may be TATAAGT or
TTTAATG (G being the -6 position and the last T the -12 posiiton) . Second is the -35 region
(A-T rich DNA sequence) , also called the Hogness TATA box ,which stretches from
position -25 to -35 . In bacteria the sequence may be TTTACA and in eukaryotes the
sequence may be TATAAAAG. Role of these regions is enhancing transcription efficiency
by increasing promoter strength – that is , the rate at which synthesis of mRNA is initiated.
The number of nucleotides that separates the two regions (-10 and -35) is also important in
determining the strength of the promoter. Optimum spacing has been found to be between 16
and 19 nucleotides.

Fig : The regions of DNA in the lac operon of E coli involved in the control of transcription

The transcription enzyme : RNA polymerase

RNA polymerase is an enzyme that produces RNA.

In prokaryotes a single RNA polymerase transcribes all the three types of RNA- rRNA
,mRNA and tRNA. In eukaryotes three RNA polymerases have been discovered – pol I and
pol III transcribe rRNA and tRNA and pol II is responsible for mRNA synthesis.

It is a multi-subunit enzyme. The functional holoenzyme is a protein complex of mw 500 kDa

consisting of two α subunits that are identical (each of mw 39 kDa) ,two β subunits that are
not identical one being 155 kDa and the other 165 kDa and a protein factor of mw 95 kDa
called σ factor. The holoenzyme is thus represented as σσββ’σ. The core enzyme is the
holoenzyme without the sigma factor – that is σσββ’. In addition ,to these components
,another protein factor called ω factor is also associated with the enzyme but its function is as
yet unknown.
 Fig : Prokaryotic RNA polymerase

Transcription Initiation :

The sigma factor of RNA polymerase recognizes the correct promoter site for the enzyme to
bind and to initiate transcription. The promoter determines which strand will be transcribed
,by virtue of the nucleotide sequence of the promoter region of the coding strand . At first the
RNA polymerase holoenzyme binds with the -35 region to form a complex which is referred
to as closed complex because the DNA at this point is still a duplex. The enzyme then moves
on towards the -10 region slowly unwinding the duplex DNA and forming single-stranded
region , the open complex , at which position transcription starts with the addition of the first
nucleotide. The first nucleotide is usually a purine , ATP or GTP. With the initiation of
transcription and after the addition of a few more nucleotides to the 3’ end , sigma factor
dissociates from the enzyme and finds another initiation site to start synthesis of the second
mRNA molecule. The release of the sigma factor relieves the tight binding of the enzyme to
the DNA template and allows free movement of the enzyme along the template . The sigma
factor thus appears to be an allosteric effector molecule in the multi-component enzyme.

Promoter selectivity is an important aspect of transcription. Since an organism has many

genes to transcribe and different genes are expressed at different times one might wonder
how the cellular transcription apparatus is modulated to affect transcription of the larger

number of genes. Recent studies indicate that the factor recognizes the promoter at two

hexanucleotide sequences located near the -35 and -10 positions relative to the site of

transcription initiation. The core enzyme without the factor is capable of initiating

transcription, but it does so randomly at many different sites in the DNA. The factor is thus

responsible for orderly transcription initiation – that is ,it initiates transcription at specific
sites in the promoter. In E coli , the factor occurs as seven different molecular species each

with ability to select specific sites of promoter. The different molecular species of factor

present in the cell serve expression of different genes at different times. The main sigma

70 54 32 28 70
factors in E coli are , and of which is present in abundance while the others

are in low concentration By increasing the concentration of the other variants under different
conditions ,the polymerase can recognize other promoter consensus sequences and thus cause
differential gene expression.

The promoter selection of RNA polymerase is further modulated by a large number of

proteins and nucleotide factors together are called transcription factors (TFs). Altogether
there are over 100 TFs in the E coli cell. The TFs are again classified into four TF groups
depending on which of the four subunits of the core enzyme they attach to at the initiation
site. These different components combine randomly to make different structural types of the
transcription apparatus- the RNA polymerase-TF complex – that can effectively transcribe all
the different genes of the organism expressed at different times and under different conditions
of growth. The 4000 or so genes in E coli for example , can thus be adequately recognized by
these different species of transcription apparatus.

Elongation :

After transcription has been accomplished , the RNA chain elongates in the 5’-3’ direction by
adding complementary ribonucleotides with uracil in place of thymine, until RNA
polymerase reaches a site on the DNA called a terminator. At this site , RNA polymerase and
the newly synthesized RNA transcript are released from the DNA.

Termination :

Several mechanisms of terminating transcription have been discovered. RNA polymerase

itself plays a role in the two principal mechanisms of transcription termination that occur in

E. coli. A cellular protein factor called rho ( ), is required in one mechanism but not the

other. These mechanisms are commonly referred to as Rho-independent and Rho-dependent

termination.
In Rho-dependent termination, the protein binds to the growing RNA strand closely
following the RNA polymerase .When the RNA polymerase encounters a termination signal
in the DNA ,the protein facilitates release of the RNA molecule from the polymerase thereby
terminating transcription.

Whereas in Rho-independent termination , the growing RNA strand is found to contain a G-C
rich region which can fold back to form hair-pin loop structure. The loop structure terminates
with a series of uridine residues. This structure works like a comma , causing the RNA
polymerase to stop for about 60 seconds and causing the mRNA transcript to fall off from the
DNA.

Fig : Transcription termination

Processing of RNA :

There are three types of RNA in the cell – ribosomal RNA (rRNA) , transfer RNA (tRNA)
and messenger (mRNA).
Ribosomal RNA

The bulk of cellular RNA is ribosomal RNA , about 80% of total RNA. The three species of
rRNA found in eukaryotes occur in the size of 28S (5 kb) ,18S (1.9 kb) and 5.8S (0.16kb).

S is the sedimentation coefficient used to indicate size of macromolecules and is determined

by the rate of the molecule during centrifugation through a density gradient. The S value
relates to molecular weight (M) by the formula :

S = 2.7 + 0.157M 0.445

In prokaryotes , the values for the corresponding species are 23S , 16S and 5S. Molecular
length of these species are 2.9 kb , 1.5 kb and 0.12 kb. In bacteria , ribosomal RNA genes are
located in an operon called rrn operon which is a 7 kb DNA segment containing the genes in
the order 5’- 16S-23S-5S-3’.In eukaryotes organization of the ribosomal RNA genes appears
to be more complex where the DNA coding for the 28S and 18S species of rRNA is located
within the nucleolar organizer region each occurring in large number of copies , over 100
,whereas genes for the 5.8S species which also occur as over 1000 tandemly duplicated
copies are interspersed within the genome separated by spacer DNA.

In bacteria , the primary rRNA transcript is a 30S precursor which undergoes post-
transcriptional cleavage and processing to form functional subunits. This involves loss of a
substantial stretch of internal nucleotides called intron or intervening sequence lying between
regions that are not lost during RNA processing , the exon. The precursor is first cut into
three fragments by endonuclease cleavage which finally mature into the 23S ,16S and 5S
rRNA species by exonuclease trimming. In eukaryotes , the primary rRNA transcript is a
45S precursor from where the three rRNA species – 28S ,18S and 5.8S are produced by
intron removal followed by splicing of the exon ends.
 Fig : Processing of rRNA in prokaryotes and eukaryotes

Transfer RNA

Transfer RNA (tRNA) is a small(4S) RNA molecule (usually about 74-95 nucleotides) that
transfers a specific active amino acid to a growing polypeptide chain at the ribosomal site of
protein synthesis during translation. About 15% of total RNA is tRNA.

Transfer RNA molecules are synthesized from DNA sequences scattered in the genome in
many copies – between 200-400. The precursors of tRNA ,pre-tRNA molecules are also
larger than mature tRNA and often contain several individual tRNA sequences. Extra bases at
the 5’ end of the pre-tRNA molecule is removed by the action of RNase P which is a complex
of RNA and protein ,but only the RNA component is sufficient to catalyze the cleavage ;
tyr (
species of RNA with such catalytic activity is called ribozyme. The pre-tRNA tRNA for
the amino acid tyrosine ) is synthesized with 128 nucleotides. During processing the extra 43
nulceotides are lost from the 5’ end so that that final product is 85 nucleotides long.
The 3’ end of tRNA is modified by RNase to terminate the end with CCA sequence which is
common to all tRNAs. In many tRNAs the CCA is transcribed from DNA ,but in some it is
added after synthesis by an enzymatic process. Another important modification after
synthesis of tRNA is modification of as many as 10% of the bases.

Structure

Transfer RNA is an adapter molecule which is able to interact both with amino acids and with
nucleic acid codons thus serving as essential interpreter in the process of translation. tRNA
molecules contain a number of unusual bases (eg dihydrouridine , pseudouridine, inosine,
lysidine) . The function of these unusual bases is not known but it is believed that since these
unusual bases cannot readily participate in conventional base pairing , they may play a role in
modifying the folded configuration of the molecule in order to render it more suitable to
correctly align to ribosome and to accept amino acids. The molecule when its structure was
fully worked out by matching the regions of complementary bases , assumed the shape of a
clover leaf as shown in fig. All tRNA molecules carry a consensus terminus 5’-CCA 3’ at the
3’-OH end of the molecule and three stem-loop regions formed by base pairing between
complementary sequences present in the molecule. The 3’-OH end of tRNA accepts amino
acid molecules and is called the amino-acyl end. One loop contains a 3 base region called the
anticodon that can base pair to the corresponding three base codon region on mRNA. The
molecule thus serves as the bridge between mRNA on the one hand and protein on the other
by being complementary to mRNA codon with its anticodon and by carrying an amino acid
by its 3’-OH end.
 Fig : Clover leaf structure of tRNA

Messenger RNA

Messenger RNA (mRNA) is a molecule of RNA encoding a chemical "blueprint" for a

protein product. mRNA is transcribed from a DNA template, and carries coding information
to the sites of protein synthesis: the ribosomes. In mRNA as in DNA, genetic information is
encoded in the sequence of nucleotides arranged into codons consisting of three bases each.

Since bacteria do not have a nucleus , the mRNA so formed do not have to cross any
membrane barrier to travel to cytoplasm in order to interact with cell’s translation
machinery , the ribosome. Thus , bacterial mRNA is accessible to the translation apparatus as
it is being formed.

Precursor mRNA (pre-mRNA) is an immature single strand of messenger ribonucleic acid

(mRNA). The pre-mRNA molecule undergoes three main modifications. These modifications
are 5' capping, 3' polyadenylation, and RNA splicing, which occur before the RNA is
translated.

Capping of the pre-mRNA involves the addition of a modified GTP , that is , a 7-methyl
guanosine triphosphate called the mGTP cap to the 5' end using a 5'-5'phosphodiester bond
, rather than the usual 5’-3’ bond.
The mGTP cap helps in proper alignment of the mRNA molecule to the ribosome during
initiation of translation. Adjacent to the cap lies a leader sequence ,also called Shine-
Dalgarno sequence (5’-AGGAGGU-3’) of 7 nulceotides which is not translated but
represents translation initiation region of the mRNA molecule. The leader sequence facilitates
binding of the ribosome to the mRNA as the first step in the complex translation process.
Bacterial mRNA does not contain poly A (a long sequence of adenine nucleotides) at the 3’
end.

Fig : Salient features of processing of bacterial and eukaryotic mRNA

Eukaryotic mRNA is in some interesting ways different from bacterial mRNA. These
molecules are synthesized inside the nucleus, but their site of action is the cytoplasm. So,
these molecules have to go through an important transport step from nucleus to the cytoplasm
across the barrier of the nuclear membrane.

The precursor eukaryotic mRNA species is synthesized within the nucleus as a class of
molecules that are highly heterogeneous in size , called heterogenous nuclear RNA or
hnRNA. A 5’ mGTP cap is added to the 5’ end similar to bacterial mRNA but unlike
bacterial mRNA .Polyadenylation involves addition of about 200 adenosine residues called
poly A tail to the 3’-OH end of the molecule. The polyadenylation process starts from the
polyadenylation signal sequence (AAUAAA) which is present in the mRNA in the
downstream region of the mRNA molecule ,that is ,towards the 3’ end. About 10 to 30
nucleotides downstream from this sequence an enzyme called poly A polymerase adds the
200 or so adenine residues.

Furthermore ,the hnRNA contains within its structure intron sequences that occur between
exon sequences. The intron is not translated but has to be removed from the molecule before
the molecule is ready for translation as functional mRNA.

RNA Splicing is a modification of an RNA after transcription, in which introns (non-coding

sequences) are removed and exons (protein-coding sequences) are joined. The splicing
process is complex but is not mediated by enzyme-driven reactions but is accomplished by
autocatalysis aided by a class of small nuclear RNA species ,some sequences within the pre-
mRNA molecule itself and a number of cofactors. RNA with such catalytic activity is called a
ribozyme – ribonucleic acid with enzymatic activity. The mechanism of ribozyme activity
presumably involves conformational changes of the target RNA molecule which generate
enough force that would break the phosphodiester bond at specific site.

Two mechanisms of splicing have been discovered . The first one that occurs in higher
eukaryotes is relatively complex and involves the participation of a complex structure
consisting of small nuclear RNAs and protein. The second is self-splicing that requires
cofactors but no RNA and protein complexes .In contrast to mRNA processing in
eukaryotes ,processing of rRNA and transfer RNA occurs by enzymatic cleavage caused by
splicing nuclease and joining of ends by ligase.

The splicing process in eukaryotes requires the formation of a complex called spliceosome
consists of small nuclear RNAs (snRNAs) and small nuclear ribonucleoproteins (snRNPs) .
The snRNAs are of 5 types called U1 , U2 ,U4 ,U5 and U6 ranging in size from 50 to about
200 nucleotides. Spliceosome-mediated splicing of mRNA involves two steps. Both steps
involve transesterification reactions that occur between RNA nucleotides. First , cleavage at
5’ splice site (5’SS) which in fact the junction of the exon and intron. Similarly there is a 3’
SS junction. These splice sites contain specific base sequences to which snRNAs can bind.
Passing through the different snRNAs the per-mRNA molecule undergoes conformational
change such that the intron forms a loop. There is then a cleavage at 5’ splicing site with the
free end of the intron folding back to join a specific site lying downstream of the intron called
branch point . The resulting structure assumes a lariat like configuration which a
characteristic intermediate of processing of many pre-mRNAs . Cleavage at 3’ SS the follows
leading to excision of the intron as a lariat.

Fig : Processing of eukaryotic pre-mRNA through autocatalytic means in the spliceosome

The actual splicing reactions are shown in fig. The intron is hydrolyzed at G and A residues
and spliced out. The adenosine residues occur towards the 3’ end of the pre-mRNA ,while the
guanosine residue is towards the 5’ end. The adenosine residue is called branch point
adenosine. The 2’OH group of the adenosine residues attacks the phosphodiester bond p at
the 5’ splice site forming the lariat intermediate. In the second step of the splicing , the 3’ OH
group of the released 5' exon attacks the 3’ splice site thus joining the exons and releasing the
lariat.
Fig : Splicing reactions of eukaryotic mRNA
Translation

Translation is the process by which the information contained in a molecule of messenger

RNA is used to synthesize a protein . The process is called translation because ribosomes
read along a messenger RNA molecule , translating the genetic code into the amino acid
sequence.

General strategy of the process :

Translation of mRNA into protein takes place in ribosomes. The process is highly complex
and is understood only in its broad outline. During translation , the message encoded in the
mRNA must be conserved as to its beginning , correct reading and with the reading
terminated at the proper place. The base sequence in the mRNA is read from a fixed starting
point by the translation machinery of the cell and it is also read by in a sequential manner.
The sequence of bases in the mRNA determines what would be the sequence of amino acids
in the polypeptide. There are 20 important amino acids in the cell which make all the
different proteins that the cell contains. There are thousands of proteins differing from one
another at the level of primary structure ,that is , in the sequence of amino acids. The
translation machinery of the cell is endowed with properties that ensure this task to be
accomplished with precision.

In the translation process tRNA serves as an adapter , a molecule able to interact both with
the mRNA and also with the building blocks of protein , that is , the amino acids. Its structure
– stem-loop organization which contains the anticodon to interact with the codon in the
mRNA and the CCA terminal at the 3-OH of the tRNA molecule that can bind with amino
acids.

There are three stages in the process of translation ; Initiation ,elongation and termination

Initiation :

All proteins of bacteria contain the amino acid N-formylmethionine (f-Met) as the fist amino
acid in the N-terminal end for which the corresponding codon in the mRNA is AUG which
also codes for normal methionine when its position is internal. The codon AUG is the
initiation codon that signals the beginning of protein synthesis.

The first step in translation is binding of mRNA to ribosome. The 5’mGTP cap of the mRNA
appears to be necessary for binding of mRNA to the small subunit of the ribosome , the 30S
subunit in bacteria. The initiation codon AUG of mRNA is included within the 30S subunit.
At this point , the 16S rRNA also binds to the mRNA preceding the initiation codon ,that is ,
with the leader sequence or the Shine-Dalgarno sequence (AGGAGGU) by the
complementary consensus sequence present in the 16S rRNA (UCCUCCA). This step is
facilitated by a protein factor called initiation factor 3 or IF-3. The aminoacyl tRNA
corresponding to the initiation codon ,that is , f-Met tRNA now binds to GTP which is also
aided by another protein factor , initiation factor 2 or IF-2 . The complex (f-Met tRNA –
GTP) is now ready to travel to the initiation codon within the ribosomal subunit , a step that
is facilitated by protein factor IF-1. This results in the formation of a complex comprising
mRNA , 30S ribosomal subunit and f-Met tRNA-GTP. At this stage , the large ribosomal
subunit is associated with the small one to form a complete ribosome. This is accomplished
by the release of the initiation factors IF-1 , IF-2 and IF-3 from the complex whereupon the
large ribosomal subunit attaches to the complex ,the GTP is hydrolyzed and complete 70S
ribosome is constituted. The initiation complex thus contains : mRNA with its initiation
codon housed in the small 30S ribosomal subunit , the f-Met tRNA attached to the mRNA
initiation codon with proper anticodon pairing and housed within the large ribosomal
subunit .
 Fig : Translation initiation

The ribosome contains three biding sites for tRNA molecules , the A site or amino acid site ;
the P site or peptidyl site and the E site or exit site. The A site holds the new tRNA with an
amino acid attached , the p site holds the previous tRNA with the growing polypeptide
attached and E site allows the used tRNA molecules to exit from the ribosome.

Transfer RNA molecules with attached amino acids are called charged tRNA molecules.
Charged tRNA molecules carry amino acids to the ribosome. tRNA carrying the amino acid
leucine is called leucine-tRNA ,one with valine is valine-tRNA and so on.

Formation of charged tRNA molecules :

At the 3’ end of tRNA molecule ,specific amino acids are attached by a two-step reaction
.Both of these reactions are however ,catalyzed by the same enzyme ,the amino-acyl tRNA
synthetase. There are many different types of this enzyme ,each specific for one particular
amino acid. The first reaction involves activation of amino acid by ATP and the second
reaction carries out the transfer of the activated amino acid to the tRNA.

amino acid + ATP → aminoacyl-AMP + PPi

aminoacyl-AMP + tRNA → aminoacyl-tRNA + AMP

Peptide bond formation :

Once P site is occupied by f-Met tRNA ,a peptide bond is formed between the two amino
acids ,that is between formmylmethionine and the incoming amino acid ,say alanine in the A
sit

Elongation :

Peptide bond formation moves the f-Met tRNA from the P-site to the E site and leaves the
ribosome. The alanine tRNA ,occupying the A-site now carries a dipeptide-methionine-
alanine. The ribosome at this stage slides to the right (towards the 3’ end of the mRNA) so
that the tRNA carrying the dipeptide comes to occupy the P site. This movement of the
ribosome along the mRNA by one triplet is called translocation. The mechanism of
translocation is not known ,but elongation factor EF-G participates in the process in
prokaryotes while in eukaryotes the corresponding factor is eEF-2. The result of translocation
is that the A-site now falls vacant and ready to receive another charged tRNA corresponding
to the codon and the process is continued until a termination codon is encountered.
 Fig : The process of polypeptide chain elongation

Termination :

Termination occurs when one of the three termination codons (UAA , UAG or UGA) moves
into the A site. These codons are not recognized by any tRNAs. Instead, they are recognized
by proteins called release factors, namely RF1 (recognizing the UAA and UAG stop codons)
or RF2 (recognizing the UAA and UGA stop codons). These factors trigger the hydrolysis of
the ester bond in peptidyl-tRNA and the release of the newly synthesized protein from the
ribosome. In eukaryotes there is only one release factor eEF which recognizes all the three
termination codons. The two subunits of the ribosome dissociates at this point and are now
free to initiate another round of translation process.

Polyribosome or polysome.

Usually one mRNA molecule is sequentially translated by a number of ribosomes so as to

enhance the yield of protein. Because of sequential and simultaneous translation of one
mRNA molecule by a series of ribosomes the mRNA in active translation process can be
visualized under the electron microscope as a string with a number of pitcher-shaped
ribosomes attached along the length of the molecule . Such a structure is called a
polyribosome or polysome.

Genetic code

Genetic code is defined as the sequence of nitrogen bases(nucleotides) in mRNA molecule

which contains the information for the synthesis of protein molecules.

There are 4 bases in nucleic acid that have to determine , in some way ,the 20 different amino
acids of the protein. Theoretical exercise on the possible nature of the genetic code began
soon after the discovery of the DNA structure. If we assume that one nucleotide codes for one
amino acid it would account for only 4 of the 20 amino acids. If a doublet of two nucleotides
for one amino acid ,then the 4 bases in various combinations of 2 would account for 4 2 = 16
amino acids ,which is still inadequate. Thus ,a three base or triplet code was suggested which
could generate 43 = 64 triplets that would be sufficient but vastly in excess.

Degeneracy of the genetic code

There are 64 possible codons in a triplet code, of which 61 have been shown to code amino
acids. Since only 20 amino acids take part in protein synthesis, it is obvious that there are
many more codons than amino acid types. Except for tryptophan and methionine, which have
a single codon each, all other amino acids involved in protein synthesis have more than one
codon. Leucine, arginine and serine have six codons each. Isoleucine has three codons. The
other amino acids each have either 2 or 4 codons each. The occurrence of more than one
codon per amino acid is called degeneracy.

The degeneracy of the code is not random. Usually the multiple codons specifying an amino
acid differ by only one base ,the third base of the codon which could be any one of the four
bases and yet the codon will specify the same amino acid . Thus ,proline is specified by the
triplets CCU ,CCA ,CCG and CCC.

Wobble Hypothesis
The triplet code is a degenerate means it lacks specificity and one amino acid often has more
than one codon. An explanation for this degeneracy is provided by the 'wobble hypothesis'
proposed by Crick (1966). Since there are 61 codons specifying amino acids, the cell should
contain 61 different tRNA molecules, each with a different anticodon . Actually, however,
the number of tRNA molecule types discovered is much less than 61. This implies that the
anticodon of some tRNAs read more than one codon on mRNA. Crick suggested that once
the first two positions of a triplet are fixed the third position pairing need not be entirely
stringent and some degree of wobbling may be permitted in its pairing with the corresponding
base in the tRNA anticodon. Thus a single tRNA is able to recognize two or more codons
differing only in the third base.

For example, the anticodon UCG of serine tRNA recognizes the codon AGC  in the mRNA.
Here the third base G of tRNA pairs with C of mRNA. G-C pairing is the normal base
pairing. The UCG anticodon can also pair with another codon AGU. Here the third base G of
tRNA pairs with U of mRNA.

Characteristics of Genetic Code

The genetic code has the following characteristics:

 The genetic code is a triplet code : Three adjacent bases, termed as codon , specify
one amino acid. For example UUU specifies phenylalanine
 Non-overlapping : Adjacent codons do not overlap that is, the sequence GCCCAC
contains two triplets, “GCC” and “CAC” not counting the “CCC” and other
subsequent three-letter sequences.
 No punctuation: The genetic code is comma less.
 The genetic code is universal i.e., a given codon specifies the same amino acid in all
organisms (with minor exceptions)
 The genetic code is degenerate ; it lacks specificity and one amino acid often has
more than one code triplet. For example, three amino acids arginine, serine and
leucine are each specified by six different codons.
 Three of the 64 codons, names UAA, UAG and UGA do not specify any amino acid
but signal the end of the message. They are called nonsense or termination codons.
 AUG codon is called starting or chain initiation codon because it initiates the
synthesis of polypeptide chain.
 \

Genetic code determination / Deciphering the genetic code

The first genetic code was identified by Nirenberg and Mathaei in 1961 at the National
Institutes of Health and the first codon identified was UUU. This code determines the amino
acid Phenylalanine.

They used a cell-free system which consisted of cell extracts containing all the necessary
components for protein synthesis to translate a synthetic mRNA called poly U (i.e.,
UUUUU...) and discovered that the polypeptide that they had synthesized consisted of only
the amino acid phenylalanine. They thereby deduced that the codon UUU specified the amino
acid phenylalanine. This was followed by experiments in the laboratory of Severo Ochoa
demonstrating that the poly-adenine RNA sequence (AAAAA...) coded for the polypeptide,
poly-lysine. and the poly-cytosine RNA sequence (CCCCC...) coded for the polypeptide,
poly-proline. Therefore, the codon AAA specified the amino acid lysine, and the codon CCC
specified the amino acid proline. Using different copolymers most of the remaining codons
were then determined.

Khorana synthesized heteropolymers having known sequences using two types of

nucleotides, namely C and U. By combining C and U a dinucleotide CU is formed.
Combining two such dinucleotide tetranucleotide (CUCU) is produced. Adding such
nucleotides results in the synthesis of the polynucleotide CUCUCUCUCU…. This
polynucleotide contains alternating CUC and UCU codons. When this polynucleotide is used
for protein synthesis, a polypeptide chain containing alternating units of amino acids leucine
and serine is produced. Thus, it is proved that the codon CUC codes for leucine and UCU
codes for serine.

More precise assignments were possible by using an assay system where synthetic triplets
were allowed to bind to ribosome in similar cell free systems and then by monitoring which
amino acids is taken up by the ribosome corresponding to a given triplet , that is , an assay of
binding of an amino acid to ribosome stimulated by specific triplets. Thus , in a short time the
deduced genetic code was experimentally validated.

The three triplets – UAA ,UAG and UGA – when tested in vitro using the above technique of
amino acid binding to ribosome , were found to be inactive ; these triplets did not stimulate
binding of any one of the 20 amino acids but was later found to be performing an essential
role as termination codons in protein synthesis.
Mutation

Mutations are changes in the DNA sequence of a cell's genome and are caused by radiation,
viruses, transposons and mutagenic chemicals, as well as errors that occur during meiosis or
DNA replication

There are two main classes of mutations : base-substitution and frameshift mutation

Base-substitution mutations occur when a single nucleotide replaces another in the DNA
sequence. Base substitution mutations can be further divided into silent mutation , missense
mutations and non-sense mutations.

If the base substitution produces no change in the amino acid sequence of the resulting
protein it is a silent mutation. Since there is no change in the amino acid sequence of the
resulting protein , silent mutations affect the genotype of the organism but not the phenotype.

If a base-substitution mutation causes a single amino acid to change in the protein , the
mutation is the missense mutation. Depending on the nature of the amino acid substitution
,the missense mutation can be harmful ,neutral or even beneficial in rare cases.

If the base substitution causes a codon to change from one coding for an amino acid to a stop
codon , it is a non-sense mutation. In general , non-sense mutations are harmful since they
lead to the premature termination of the protein.

If nucleotides are inserted into or removed from the DNA sequence ,the resulting mutation is
known as a frameshift mutation. Because the ribosome translates mRNA into protein by
reading the mRNA in three nucleotide codons , if one or two nucleotides are removed and
inserted , the result is a shift in the reading frame of the codons. This leads to a change in
nearly every codon and nearly every amino acid that follows the site of the frameshift
mutation . Frameshift mutations usually lead to a nonfunctional protein.

If one or more bases are added to a DNA sequence causing a shift in the reading frame of the
resulting codons , the mutation is a frameshift insertion.

If one or more bases are removed from a DNA sequence causing a shift in the reading frame
of the resulting codons , the mutation is a frameshift deletion.

A suppressor mutation is a mutation that counteracts the phenotypic effects of another

mutation.

There are several classes

‘Intragenic suppression’ results from a second mutation that corrects the functioning of the
mutant gene (e.g. a mutation of a different nucleotide in the same triplet, such that the codon
then encodes the original amino acid);
‘Intergenic suppression’ results from mutation of a different gene, the product of which
compensates for the dysfunction in the first (e.g. a mutation that produces a mutant transfer-
RNA molecule that inserts an amino acid in response to a nonsense codon, thus continuing a
protein that would otherwise have been terminated).

If a single suppressor mutation can suppress more than one existing mutation, it is said to be a
supersuppressor.

N:B : Mutations that change only one base pair are called point mutations. Point mutations
are caused by base-pair substitutions in the DNA