■ Genetic information is stored in DNA by means of a triplet code that

is nearly universal to all living things on Earth.

■ The genetic code is initially transferred from DNA to RNA, in the
process of transcription.
■ Once transferred to RNA, the genetic code exists as triplet codons,
which are sets of three nucleotides in which each nucleotide is one of
the four kinds of ribonucleotides composing RNA.
■ RNA’s four ribonucleotides, analogous to an alphabet of four
“letters,” can be arranged into 64 different three-letter sequences.
Most of the triplets in RNA encode one of the 20 amino acids present
in proteins, which are the end products of most genes.
■ Several codons act as signals that initiate or terminate protein
synthesis.
■ In eukaryotes, the process of transcription is similar to, but more
complex than, that in prokaryotes and in the bacteriophages that
infect them.
The Genetic Code Uses Ribonucleotide Bases as “Letters”
1. The genetic code is written in linear form, using as “letters” the
ribonucleotide bases that compose mRNA molecules.
2. Each “word” within the mRNA consists of three ribonucleotide
letters, thus referred to as a triplet code. With several exceptions, each
group of three ribonucleotides, called a codon, specifies one amino
acid.
The Genetic Code Uses Ribonucleotide Bases as “Letters”
3. The code is unambiguous—each triplet specifies only a single amino
acid.
4. The code is degenerate, meaning that a given amino acid can be
specified by more than one triplet codon.
This is the case for 18 of the 20 amino acids.
The Genetic Code Uses Ribonucleotide Bases as “Letters”
5. The code contains one “start” and three “stop” signals, triplets that
initiate and terminate translation, respectively.
6. Once translation of mRNA begins, the codons are read one after the
other with no breaks between them (until a stop signal is reached).
7. The code is nearly universal. With only minor exceptions, a single
coding dictionary is used by almost all viruses, prokaryotes, archaea,
and eukaryotes.
Information present on one of the two
strands of DNA (the template strand) is
transferred into an RNA complement
through the process of transcription.
Once synthesized, this RNA acts as a
“messenger” molecule, transporting the
coded information out of the nucleus—
hence its name, messenger RNA
(mRNA). The mRNAs then associate
with ribosomes, where decoding into
proteins occurs.
The Triplet Nature of the Code

The effect of frameshift mutations

on a DNA sequence repeating the
triplet sequence GAG.
(a) The insertion of a single
nucleotide shifts all subsequent
triplets out of the reading frame.
(b) The insertion of three
nucleotides changes only two
triplets, after which the original
reading frame is reestablished.
 The Triplet Nature of the Code

A code of four nucleotides combined into two-letter words, for

example, provides only 16 unique code words (42).
A triplet code yields 64 words (43) and therefore is sufficient for
the 20 needed.
 Degeneracy
Almost all amino acids are specified by two, three, or four different
codons.
Three amino acids (serine, arginine, and leucine) are each encoded
by six different codons.
Only tryptophan and methionine are encoded by single codons.
 Wobble hypothesis

Crick’s hypothesis predicted that the initial two ribonucleotides of

triplet codes are often more critical than the third member in
attracting the correct tRNA.

He hypothesised that the hydrogen bonding occurring in the third

position of the codon-anticodon interaction would be less restricted
in space and may not have to strictly follow the predetermined rules
of base-pairing.
This relaxed base-pairing requirement, or “wobble,” allows the
anticodon of a single form of tRNA to pair with more than one triplet
in mRNA.

As a result of these wobble rules, only about 30 different tRNA

species are necessary to accommodate the 61 codons specifying an
amino acid.
 The Ordered Nature of the Code
Chemically similar amino acids often share one or two “middle” bases
in the different triplets encoding them.
For example, either U or C is often present in the second position of
triplets that specify hydrophobic amino acids, including valine and
alanine, among others.
 What advantage does this feature provide?
The end result of an “ordered” code is that it buffers the potential
effect of mutation on protein function. While many mutations of the
second base of triplet codons result in a change of one amino acid to
another, the change is often to an amino acid with similar chemical
properties. In such cases, protein function may not be noticeably
altered.
 Initiation, Termination and Suppression

Only one codon, AUG, codes for methionine, and it is

sometimes called the initiator codon.
Three other codons (UAG, UAA, and UGA) serve as
termination codons, punctuation signals that do not code for
any amino acid. They are not recognized by a tRNA molecule,
and translation terminates when they are encountered.
 Initiation, Termination and Suppression

Mutations that produce any of the three codons internally in a

gene also result in termination.
In that case, only a partial polypeptide is synthesized, since it is
prematurely released from the ribosome.
When such a change occurs in the DNA, it is called a nonsense
mutation.
 The Genetic Code is Nearly Universal
The genetic code would be found to be universal, applying equally
to viruses, bacteria, archaea, and eukaryotes.
Many recent studies involving recombinant DNA technology reveal
that eukaryotic genes can be inserted into bacterial cells, which are
then transcribed and translated.
 Different Initiation Points Create
Overlapping Genes

Single mRNA may have multiple initiation points for translation.

These points could theoretically create several different reading
frames within the same mRNA, thus specifying more than one
polypeptide.
A similar situation has been observed in other viruses and bacteria.
The employment of overlapping reading frames optimizes the limited
amount of DNA present.

The relative positions of the sequences encoding seven polypeptides of the

phage fX174.
However, such an approach to storing information has a distinct
disadvantage in that a single mutation may affect more than one
protein and thus increase the chances that the change will be
deleterious or lethal.
A single mutation in the middle of the B gene could potentially affect
three other proteins (the A, A’ and K proteins).
 Transcription Synthesizes RNA
on a DNA Template

Proteins were the end products of many genes.

▪ The complex, multistep process begins with the transfer of
genetic information stored in DNA to RNA.
▪ The process by which RNA molecules are synthesized on a DNA
template is called transcription.
 Information Flow

1. DNA is, for the most part, associated with chromosomes in the
nucleus of the eukaryotic cell. However, protein synthesis occurs in
association with ribosomes located outside the nucleus, in the
cytoplasm. Therefore, DNA does not appear to participate directly in
protein synthesis.
 Information Flow

2. RNA is synthesized in the nucleus of eukaryotic cells, in which DNA

is found, and is chemically similar to DNA.
3. Following its synthesis, most RNA migrates to the cytoplasm, in
which protein synthesis (translation) occurs.
4. The amount of RNA is generally proportional to the amount of
protein in a cell.
 RNA Polymerase Directs RNA Synthesis

Unlike DNA polymerase, no primer is required to initiate synthesis

of RNA on a DNA template.
Nucleotides are linked during synthesis by 5’ to 3’ phosphodiester
bonds.
The energy created by cleaving the triphosphate precursor into the
monophosphate form drives the reaction.
 Promoters, Template Binding and the Sigma (σ) Subunit

Transcription results in the synthesis of a single-stranded RNA

molecule complementary to a region along only one of the two
strands of the DNA double helix.
The DNA strand that is transcribed the template strand and its
complement the partner strand.
The early stages of transcription in prokaryotes
 Promoters, Template Binding and the Sigma (σ) Subunit

In bacteria, the site of this initial binding is established when the RNA
polymerase σ subunit recognizes specific DNA sequences called
promoters.
These sequences are located in the 5’ region, upstream from the
point of initial transcription of a gene.
It is believed that the enzyme “explores” a length of DNA until it encounters
a promoter region and binds there to about 60 nucleotide pairs along the
helix.
Once this occurs, the helix is denatured, or unwound, locally, making the
template strand of the DNA accessible to the action of the enzyme. The
point at which transcription actually begins is called the transcription start
site.
 Consensus sequences
Sequences that are similar (homologous) in different genes of
the same organism or in one or more genes of related organisms.
Two consensus sequences have been found in bacterial promoters.
1. TATAAT, is located 10 nucleotides upstream from the site of initial
transcription
(the -10 region, or Pribnow box).
2. TTGACA, is located 35 nucleotides upstream
(the -35 region).
Sequences such as these, in regions adjacent to the gene itself, are
said to be cis-acting elements. The term cis, drawn from organic
chemistry nomenclature, means “next to” or on the same side as
other functional groups, in contrast to being trans to or “across
from,” them.
In molecular genetics, then, cis-elements are those that are located
on the same DNA molecule. In contrast, trans-acting factors are
molecules that bind to these DNA elements.
 Initiation, Elongation, and Termination
of RNA Synthesis
Once RNA polymerase has recognized and bound to the promoter,
DNA is converted from its double-stranded form to an open
structure, exposing the template strand.
The enzyme then proceeds to initiate RNA synthesis.
Subsequent ribonucleotide complements are inserted and linked
together by phosphodiester bonds as RNA polymerization proceeds.
After these ribonucleotides have been added to the
growing RNA chain, the sigma subunit dissociates from the
holoenzyme, and chain elongation proceeds under the
direction of the core enzyme.
The unique sequence of
nucleotides in termination region
causes the newly formed
transcript to fold back on itself,
forming what is called a hairpin
secondary structure, held
together by hydrogen bonds.
 Polycistronic mRNA

In bacteria, groups of genes whose protein products are involved in

the same metabolic pathway are often clustered together along the
chromosome.
In many such cases, the genes are contiguous, and all but the last
gene lack the encoded signals for termination of transcription.
In eukaryotes, monocistronic mRNAs are the rule, although an
increasing number of exceptions are being reported.
The result is that during transcription, a large mRNA is produced,
encoding more than one protein.
The RNA is called a polycistronic mRNA.
 Transcription in Eukaryotes Differs from Prokaryotic
Transcription in Several Ways

1. Transcription in eukaryotes occurs within the nucleus under the

direction of three separate forms of RNA polymerase.
For the mRNA to be translated, it must move out of the nucleus into
the cytoplasm.
 Transcription in Eukaryotes Differs from Prokaryotic
Transcription in Several Ways

2. Initiation of transcription of eukaryotic genes requires the

compact chromatin fiber, characterized by nucleosome coiling, to be
uncoiled and the DNA to be made accessible to RNA polymerase
and other regulatory proteins (chromatin remodeling).
 Transcription in Eukaryotes Differs from Prokaryotic
Transcription in Several Ways

3. Initiation and regulation of transcription require a more extensive

interaction between cis-acting DNA sequences and trans-acting
protein factors involved in stimulating and initiating transcription.

4. Alteration of the primary RNA transcript to produce mature

eukaryotic mRNA involves many complex stages referred to generally
as “processing.”
 Heterogeneous nuclear RNA (hnRNA)

The initial (or primary) transcripts are most often much larger than
those that are eventually translated into protein. Sometimes called
pre-mRNAs, these primary transcripts are found only in the nucleus
and referred to collectively as heterogeneous nuclear RNA (hnRNA).
 Initiation of Transcription in Eukaryotes

Eukaryotic RNA polymerase exists in three distinct forms.

Each enzyme is larger and more complex than the single prokaryotic
polymerase.
For example, in yeast, the holoenzyme consists of two large subunits
and 10 smaller subunits.
 Initiation of Transcription in Eukaryotes
RNA polymerase II (RNP II), which is responsible for the transcription
of a wide range of genes in eukaryotes.
The activity of RNP II is dependent on both cis-acting elements
surrounding the gene itself and a number of trans-acting transcription
factors that bind to these DNA elements.
 Initiation of Transcription in Eukaryotes

At least four cis-acting DNA elements regulate the initiation of

transcription by RNP II.
The first of these elements, the core-promoter, determines where
RNP II binds to the DNA and where it begins copying the DNA into
RNA.
 Initiation of Transcription in Eukaryotes

The other three types of regulatory DNA sequences, called proximal-

promoter elements, enhancers, and silencers, influence the efficiency
or the rate of transcription initiation by RNP II.

In many eukaryotic genes, a cis-acting core-promoter element is

the TATA box.
 Enhancers and Silencers

Enhancers increase transcription levels and silencers decrease them.

The locations of these elements can vary from immediately upstream
from a promoter to downstream, within, or kilobases away, from a
gene.
Each eukaryotic gene has its own unique arrangement of
proximalpromoter, enhancer, and silencer elements.
 Transcription Factors

Complementing the cis-acting regulatory sequencesare various trans-

acting factors that facilitate RNP II binding and, therefore, the
initiation of transcription.
These proteins are referred to as transcription factors.
 Transcription Factors

There are two broad categories of transcription factors:

1. The general transcription factors (GTFs) that are absolutely
required for all RNP II–mediated transcription.
2. The transcriptional activators and repressors that influence the
efficiency or the rate of RNP II transcription initiation.
 Transcription Factors

The general transcription factors are essential because RNP II cannot

bind directly to eukaryotic core-promoter sites and initiate
transcription without their presence.
The general transcription factors involved with human RNP II binding
are well characterized and designated TFIIA, TFIIB, and so on.
 Transcription Factors

One of these, TFIID, binds directly to the TATA-box sequence. Once

initial binding of TFIID to DNA occurs, the other general transcription
factors, along with RNP II, bind sequentially to TFIID, forming an
extensive preinitiation complex.
 Transcription Factors

The specific transcription factors (activators and represssors) bind to

enhancer and silencer elements and regulate transcription initiation
by aiding or preventing the assembly of pre-initiation complexes.
 Elongation and Termination

 Elongation works similarly to prokaryotes.

 At the end of transcription, the part in the DNA that carries the
termination signal is reached.
 The complex becomes unstable.
 The clamp opens, transcription ends and DNA and RNA are
separated from the enzyme.
 Heterogeneous Nuclear RNA and Its
Processing: Caps and Tails
Eukaryotic RNA transcripts require significant alteration before they
are transported to the cytoplasm and translated.
The initial transcript of a gene results in a large RNA molecule that
must be processed in the nucleus before it appears in the cytoplasm as
a mature mRNA molecule.
 Post Transcriptional Modifications
Transcription produces a pre-mRNA containing a leader sequence
(L), several introns (I), and several exons (E), as identified in the
DNA template strand. This is processed by the addition of a 5’7-
mG cap and a 3’-poly-A tail.
The introns are then spliced out and the exons joined to create
the mature mRNA.
Poly A has also been found at the 3’ end of almost all mRNAs in a variety of
eukaryotic organisms.
 Post Transcriptional Modifications
A 7-methylguanosine (7-mG) cap is added even before synthesis of the
initial transcript is complete and appears to be important to
subsequent processing within the nucleus.
The cap also protects the 5’ end of the molecule from nuclease attack.
 The Coding Regions of Eukaryotic Genes Are Interrupted
By Intervening Sequences

Internal DNA sequences are represented in initial RNA transcripts, but

they are removed before the mature mRNA is translated.
Such nucleotide segments are called intervening sequences, and the
genes that contain them are split genes. DNA sequences that are not
represented in the final mRNA product are also called introns (“int”
for intervening), and those retained and expressed are called exons
(“ex” for expressed).
 Splicing Mechanisms

The discovery of split genes led to intensive attempts to elucidate

the mechanism by which introns of RNA are excised and exons
are spliced back together.
A specific endonuclease recognizes the intron termini and
excises the intervening sequences. Then RNA ligase seals the
exon ends to complete each splicing event.
 Splicing Mechanisms:
Self-Splicing RNAs
Introns in eukaryotes can be categorized into several groups
based on their splicing mechanisms.
Group I splicing mechansim require no additional components
for intron excision; the intron itself is the source of the enzymatic
activity necessary for removal.
RNAs that are capable of catalytic activity are referred to as
ribozymes.
 Splicing Mechanisms:
Self-Splicing RNAs
Self-excision also seems to govern the removal of introns from
the primary mRNA and tRNA transcripts produced in
mitochondria and chloroplasts. These are referred to as group II
introns.
As in group I molecules, splicing here involves two autocatalytic
reactions leading to the excision of introns. However, guanosine
is not involved as a cofactor with group II introns.
 Splicing Mechanisms: The Spliceosome

Compared to the group I and group II introns discussed above,

those in nuclear-derived mRNA can be much larger—up to
20,000 nucleotides—and they are more plentiful. Their removal
appears to require a much more complex mechanism.
 Splicing Mechanisms: The Spliceosome

The splicing reactions are mediated by a huge molecular complex

called a spliceosome.
One set of essential components of spliceosomes is a unique set of
small nuclear RNAs (snRNAs). These RNAs are usually 100 to 200
nucleotides long or less and are complexed with proteins to form
small nuclear ribonucleoproteins (snRNPs or snurps). Because they
are rich in uridine residues, the snRNAs have been arbitrarily
designated U1, U2, . . ., U6.
Excision is dependent on various snRNAs
(U1, U2, . . ., U6) that combine with
proteins to form snRNPs, which function
as part of a large structure referred to as
the spliceosome. The lariat structure in
the intermediate stage is characteristic of
this mechanism.
 Splicing Mechanisms: The Spliceosome

Several cases are known wherein introns present in pre-mRNAs

derived from the same gene are spliced in more than one way, thereby
yielding different collections of exons in the mature mRNA.
Such alternative splicing yields a group of similar but nonidentical
mRNAs that, upon translation, result in a series of related proteins
called isoforms.
 Alternative Splicing

Many examples have been encountered in organisms ranging from

viruses to Drosophila to humans.
Alternative splicing of pre-mRNAs represents a way of producing
related proteins from a single gene, increasing the number of gene
products that can be derived from an organism’s genome.