UNIT – III – TRANSCRIPTION

RIBONUCLEIC ACID

 RNA is a polymer of ribonucleotides linked together by 3’-5’ phosphodiester linkage.

 The major role of RNA is to participate in protein synthesis, which requires three classes of
RNA.

STRUCTURE OF RNA

 Back bone is sugar and phosphate group

 Nitrogenous bases linked to sugar moiety project from the backbone

 Nitrogenous bases are linked to pentose sugar through N-glycosidic linkage to form a
nucleoside

 Phosphate group is linked with 3’OH of nucleoside through phosphoester linkage

 2 nucleotides are linked through 3’-5’-phosphodiester linkage to form a dinucleotide

 More and more such groups will be linked to form a poly nucleotide chain

 Such a polymer has a free phosphate moiety at 5’ end of ribose sugar and it is called as 5’-
end of polynucleotide chain

 At other end, ribose has free 3’-OH group which is called as the 3’-end of polynucleotide
chain

 In RNA, every nucleotide has an additional-OH present at 2’-position of ribose

  Synthesis of RNA is usually catalyzed by an enzyme—RNA polymerase

 By using DNA as a template

 The process is known as transcription

 There are also a number of RNA-dependent RNA polymerases that use RNA as their template
for synthesis of a new strand of RNA

 A number of RNA viruses (such as poliovirus) use this type of enzyme to replicate their
genetic material

BASIC PRINCIPLES OF TRANSCRIPTION AND TRANSLATION

• RNA is the bridge between genes and the proteins for which they code

• Transcription is the synthesis of RNA using information in DNA

• Transcription produces messenger RNA (mRNA)

• Translation is the synthesis of a polypeptide, using information in the mRNA

• Ribosomes are the sites of translation

• In prokaryotes, translation of mRNA can begin before transcription has finished

• In a eukaryotic cell, the nuclear envelope separates transcription from translation

• Eukaryotic RNA transcripts are modified through RNA processing to yield the finished
mRNA

• A primary transcript is the initial RNA transcript from any gene prior to processing

• The central dogma is the concept that cells are governed by a cellular chain of command:
DNA  RNA  protein

DNA RNA Protein

TYPES OF RNA

In all prokaryotic and eukaryotic organisms, three main classes of RNA molecules exist-

1) Messenger RNA(m RNA)

2) Transfer RNA (t RNA)

3) Ribosomal RNA (r RNA)

The other are –

o small nuclear RNA (SnRNA),

o micro RNA(mi RNA) and

o small interfering RNA(Si RNA) and

o heterogeneous nuclear RNA (hnRNA)

Heterogeneous nuclear RNA (hnRNA)

 In mammalian nuclei , hnRNA is the immediate product of gene transcription

 The nuclear product is heterogeneous in size (Variable) and is very large.

 Molecular weight may be more than 107, while the molecular weight of m RNA is less than
2x 106

 75 % of hnRNA is degraded in the nucleus, only 25% is processed to mature m RNA

Messenger RNA

 Messenger RNA (mRNA) carries information about a protein sequence to the ribosomes, the
protein synthesis factories in the cell

 It is coded so that every three nucleotides (a codon) correspond to one amino acid

 . In eukaryotic cells, once precursor mRNA (pre-mRNA) has been transcribed from DNA, it
is processed to mature mRNA

 This removes its introns—non-coding sections of the pre-mRNA

 The mRNA is then exported from the nucleus to the cytoplasm, where it is bound to
ribosomes and translated into its corresponding protein form with the help of tRNA
Ribosomal RNA

The mammalian ribosome contains two major nucleoprotein subunits—a larger one with a molecular
weight of 2.8 x 106 (60S) and a smaller subunit with a molecular weight of 1.4 x 106 (40S).

 Is a structural and functional component of ribosomes w/ are “platforms” on which protein

synthesis occur.

 Consist of about 35% protein and 65% ribosomal RNA.

 Complexed w/ proteins, the rRNA forms the cellular structures called the ribosomes.

 Ribosomal RNA (rRNA) is the catalytic component of the ribosomes

 Eukaryotic ribosomes contain four different rRNA molecules: 18S, 5.8S, 28S and 5S rRNA

 Three of the rRNA molecules are synthesized in the nucleolus, and one is synthesized
elsewhere.

 In the cytoplasm, ribosomal RNA and protein combine to form a nucleoprotein called a
ribosome

 The ribosome binds mRNA and carries out protein synthesis

 Several ribosomes may be attached to a single mRNA at any time.

 Nearly all the RNA found in a typical eukaryotic cell is rRNA.

Transfer RNA

1) Primary structure- The nucleotide sequence of all the t RNA molecules allows extensive intrastand
complimentarity that generates a secondary structure.

2) Secondary structure- Each single t- RNA shows extensive internal base pairing and acquires a
clover leaf like structure. The structure is stabilized by hydrogen bonding between the bases and is a
consistent feature.
  The L shaped tertiary structure is formed by further folding of the clover leaf due to hydrogen
bonds between T and D arms.

 The base paired double helical stems get arranged in to two double helical columns,
continuous and perpendicular to one another.

Small nuclear RNA (SiRNA)

 Most of these molecules are complexed with proteins to form small nuclear
ribonucleoproteins particles{snurps} and are distributed in the nucleus, in the cytoplasm, or
in both.

 They range in size from 20 to 300 nucleotides and are present in 100,000–1,000,000 copies
per cell.

Function:

 Help w/ the processing of the initial mRNA into mature form that is ready for export out of
the nucleus-{ splicing}.

 Of the several snRNAs, U1, U2, U4, U5, and U6 are involved in intron removal and the
processing of hnRNA into mRNA

 The U7 snRNA is involved in production of the correct 3' ends of histone mRNA—which
lacks a poly(A) tail.

MicroRNA, mi RNA, Small interferingRNA, siRNA

 These two classes of RNAs represent a subset of small RNAs; both play important roles in
gene regulation.

 miRNAs and siRNAs cause inhibition of gene expression by decreasing specific protein
production albeit apparently via distinct mechanisms

Five different types of RNA, each encoded by different genes:

1. mRNA Messenger RNA, encodes the amino acid sequence of a
polypeptide.

2. tRNA Transfer RNA, transports amino acids to ribosomes during

translation.

3. rRNA Ribosomal RNA, forms complexes called ribosomes with protein,

the structure on which mRNA is translated.

4. snRNA Small nuclear RNA, forms complexes with proteins used in

eukaryotic RNA processing (e.g., exon splicing and intron
removal).
5. miRNA/siRNA
Micro RNA/small interfering RNA, short ~22 nt RNA sequences
that bind to 3’ UTR target mRNAs and result in gene silencing.

Transcription

When a protein is needed by a cell, the genetic code for that protein must be read from the DNA and
processed.

A two step process:

1. Transcription = synthesis of a single-stranded RNA molecule using the DNA template (1

strand of DNA is transcribed).

2. Translation = conversion of a messenger RNA sequence into the amino acid sequence of a
polypeptide (i.e., protein synthesis)

 Both processes occur throughout the cell cycle. Transcription occurs in the nucleus, whereas
translation occurs in the cytoplasm.

Materials required

 The enzyme RNA polymerase or DNA directed RNA polymerase

 DNA template – the transcription unit

 All the four types of ribonucleoside triphosphates (ATP, CTP, GTP and UTP)
 Divalent metal ions Mg2+ or Mn2+ as a cofactor

 No primer is needed for RNA synthesis

Transcription: How is an RNA strand synthesized?

1. Regulated by gene regulatory elements within each gene.

2. DNA unwinds next to a gene.

3. RNA is transcribed 5’ to 3’ from the template (3’ to 5’).

4. Similar to DNA synthesis, except:

 NTPs instead of dNTPs (no deoxy-)

 No primer

 No proofreading

 Adds Uracil (U) instead of thymine (T)

 RNA polymerase

RNA POLYMERASE

 - Two  polypeptide chains, coded by gene rpoA, assembly of core enzyme & and help in the
probably in the recognition of promoter

β – one copy of β subunit, , coded by gene rpoB, binds with the incoming nucleotides & helps in
the formation of the first phosphodiester bond

β’ – one copy of β’ subunit, , coded by gene rpoC, binds with the template strand or antisense
DNA strand

σ - Single polypeptide chain, loosely attached to the core enzyme.

σ Subunit

• Recognises the start signal on DNA molecules and directs the core enzyme of RNA
polymerase to bind to the promoter region upstream of initiation codon

• Recognises two special sequences of bases in the promoter region of the coding strand (i.e.
Antitemplate strand) of DNA - -10 sequence and -35 sequence

• Recognises of promoter sequence, facilitates opening or melting of DNA helix

• Separates from core enzyme once about 10nt are joined to initiate RNA synthesis

FUNCTIONS OF RNA POLYMERASE

• Unwinds about 15 bases of DNA around the initiation site to form an open promoter – DNA
complex and provides single strand of DNA to act as template for transcription

• Catalyses the formation of phosphodiester bonds between successive nt of a polynucleotide

chain during synthesis of RNA

 Lacks proof reading 3’-5’ exonuclease activity

 Therefore, one error for every 104 to 105 ribonucleotides incorporated is introduced during
RNA transcription

 But the mistake during transcription is not serious because of its high turnover and Wobble
pairing during translation

Three Steps to Transcription:

1. Initiation

2. Elongation

3. Termination

 Occur in both prokaryotes and eukaryotes.

 Elongation is conserved in prokaryotes and eukaryotes.

 Initiation and termination proceed differently.

INITIATION
Each gene has three regions:

1. 5’ Promoter, attracts RNA polymerase

-10 bp 5’-TATAAT-3’

-35 bp 5’-TTGACA-3’

2. Transcribed sequence (transcript) or RNA coding sequence

3. 3’ Terminator, signals the stop point

Step 1-Initiation, E. coli model:

1. RNA polymerase combines with sigma factor (a polypeptide) to create RNA polymerase
holoenzyme

 Recognizes promoters and initiates transcription.

 Sigma factor required for efficient binding and transcription.

 Different sigma factors recognize different promoter sequences.

2. RNA polymerase holoenzyme binds promoters and untwists DNA

 Binds loosely to the -35 promoter (DNA is d.s.)

 Binds tightly to the -10 promoter and untwists

3. Different types and levels of sigma factors influence the level and dynamics of gene
expression (how much and efficiency).
Step 2-Elongation, E. coli model:

1. After 8-9 bp of RNA synthesis occurs, sigma factor is released and recycled for other
reactions.

2. RNA polymerase completes the transcription at 30-50 bp/second (and order of magnitude
slower than DNA polymerase).

3. DNA untwists rapidly, and re-anneals behind the enzyme.

4. Part of the new RNA strand is hybrid DNA-RNA, but most RNA is displaced as the helix
reforms.

Step 3-Termination, E. coli model:

Two types of terminator sequences occur in prokaryotes:

1. Type I (-independent)

Palindromic, inverse repeat forms a hairpin loop and is believed to physically destabilize the
DNA-RNA hybrid.
 2. Type II (-dependent)

Involves  factor proteins that break the hydrogen bonds between the template DNA and
RNA.

The Basic Rules of Transcription

Before we examine the process of eukaryotic transcription, let’s pause to summarize some of the
general principles of bacterial transcription.

1. Transcription is a selective process; only certain parts of the DNA are transcribed at any one time.

2. RNA is transcribed from single-stranded DNA. Within a gene, only one of the two DNA strands—
the template strand—is normally copied into RNA.

3. Ribonucleoside triphosphates are used as the substrates in RNA synthesis. Two phosphate groups
are cleaved from a ribonucleoside triphosphate, and the resulting nucleotide is joined to the 3′-OH
group of the growing RNA strand.

4. RNA molecules are antiparallel and complementary to the DNA template strand. Transcription is
always in the 5′→3′ direction, meaning that the RNA molecule grows at the 3′ end.

5. Transcription depends on RNA polymerase—a complex, multimeric enzyme. RNA polymerase

consists of a core enzyme, which is capable of synthesizing RNA, and other subunits that may join
transiently to perform additional functions.

6. A sigma factor enables the core enzyme of RNA polymerase to bind to a promoter and initiate
transcription.
7. Promoters contain short sequences crucial in the binding of RNA polymerase to DNA; these
consensus sequences are interspersed with nucleotides that play no known role in transcription.

8. RNA polymerase binds to DNA at a promoter, begins transcribing at the start site of the gene, and
ends transcription after a terminator has been transcribed.

EUKARYOTIC TRANSCRIPTION

Most eukaryotic cells

possess three distinct types of RNA polymerase, each of
which is responsible for transcribing a different class of
RNA: RNA polymerase I transcribes rRNA; RNA polymerase
II transcribes pre-mRNAs, snoRNAs, some miRNAs,
and some snRNAs; and RNA polymerase III transcribes
other small RNA molecules—specifically tRNAs, small
rRNA, some miRNAs, and some snRNAs (Table 13.3). RNA
polymerases I, II, and III are found in all eukaryotes. Two
additional RNA polymerases, RNA polymerase IV and RNA
polymerase V, have been found in plants. RNA polymerases
IV and V transcribe RNAs that play a role in DNA methylation
and chromatin structure.
All eukaryotic polymerases are large, multimeric enzymes,
typically consisting of more than a dozen subunits. Some sub- units are common to all RNA
polymerases, whereas others
are limited to one of the polymerases. As in bacterial cells, a
number of accessory proteins bind to the core enzyme and
affect its function.
A significant difference between bacterial and eukaryotic transcription is the existence of three
different eukaryotic RNA polymerases, which recognize different types of promoters. In bacterial
cells, the holoenzyme (RNA polymerase plus the sigma factor) recognizes and binds directly to
sequences in the promoter. In eukaryotic cells, promoter recognition is carried out by accessory
proteins that bind to the promoter and then recruit a specific RNA polymerase (I, II, or III) to the
promoter.
One class of accessory proteins comprises general transcription factors, which, along with RNA
polymerase, form the basal transcription apparatus—a group of proteins that assemble near the
start site and are sufficient to initiate minimal levels of transcription. Another class of accessory
proteins consists of transcriptional activator proteins, which bind to specific DNA sequences and
bring about higher levels of transcription by stimulating the assembly of the basal transcription
apparatus at the start site.
A promoter for a gene transcribed by RNA polymerase II typically consists of two primary parts: the
core
promoter and the regulatory promoter.
Core promoter The core promoter is located immediately upstream of the gene and is the site to
which the basal transcription apparatus binds. The core promoter typically includes one or more
consensus sequences. One of the most common of these sequences is the TATA box, which has the
consensus sequence TATAAA and is located from −25 to −30 bp upstream of the start site. Additional
consensus sequences that may be found in the core promoters of genes transcribed by RNA
polymerase II are shown in Figure 13.15. These consensus sequences are recognized by transcription
factors that bind to them and serve as a platform for the assembly of the basal transcription apparatus.
Regulatory promoter The regulatory promoter is located immediately upstream of the core
promoter. A variety of different consensus sequences can be found in the regulatory promoters, and
they can be mixed and matched in different combinations. Transcriptional activator proteins bind to
these sequences and either directly or indirectly make contact with the basal transcription apparatus
and affect the rate at which transcription is initiated.
Transcriptional activator proteins also regulate transcription by binding to more-distant sequences
called enhancers. The DNA between an enhancer and the promoter loops out, and so transcriptional
activator proteins bound to the enhancer can interact with the basal transcription machinery at the
core promoter.
Polymerase I and III promoters RNA polymerase I and RNA polymerase III each recognize
promoters that are distinct from those recognized by RNA polymerase II. For example, promoters
for small rRNA and tRNA genes, transcribed by RNA polymerase III, contain internal promoters
that are downstream of the start site and are transcribed into the RNA.

Initiation of Eukaryotic Transcription

For the eukaryotic transcription the regulatory DNA sequences (such as promoters, enhancers

and silencers) for genes transcribed by each of the three RNA polymerases differ. Various
transcription factors are also involved in the formation of a transcription complex which are needed
for initiation of transcription. Generally, each of RNA polymerase is believed to have its own set of
transcription factors, however, TF II D or a part of it (e.g., TBP=TATA binding protein) is required
for all the three RNA polymerases.

The transcription factors (TFs) can be defined as proteins, which are needed for initiation of
transcription, but are not part of the RNA polymerase. They help in DNA binding of a RNA
polymerase to constitute the so-called pre-initiation complex or transcription complex. After the
formation of this complex initiation of transcription occurs. All known transcription factors may
recognize either DNA sequences, another factor or RNA polymerase.

Formation of transcriptosome with RNA pol II.

A promoter sequence which is responsible for constitutive expression of common genes (also called
house keeping genes) in all cells, is called generic promoter.

The generic promoter cannot bring about regulated expression (i.e., tissue or stimulus specific
expression of genes, called luxary genes).

Initiation of transcription on the generic promoter by RNA polymerase II requires the action of
diverse transcription factors (TFs) in the following order :

(i) TF II D binds at TATA box;

(ii) the step (i) permits the association of TF IIA and TF IIB;

(iii) TF II B forms the so-called DB complex and RNA polymerase II associates to promoter site;

(iv) RNA pol II is accompanied to the promoter by TF II F to form a transcription complex ;

(v) orderly addition of TF II E, TF II H and TF II J helps the initiation process.

Elongation of RNA Chain in Eukaryotes

There are certain accessory proteins of transcription, called elongation factors, which enhance the
overall activity of RNA polymerase II and lead to increase in the elongation rate. At least two such
proteins are known:

(1) The TF II F accelerates RNA chain growth relatively uniformly in concord with RNA
polymerase II.

(2) The TF II S (also called S II) helps in elongation of RNA chain by unburdening the
obstruction in the path of such elongation. TF II S is known to act by first causing hydrolytic
cleavage at 3´ end of RNA chain, thereby, helping in the forward movement of RNA
polymerase through any block to elongation.

Termination of Eukaryotic Transcription

(1) In eukaryotes, the actual termination of RNA polymerase II activity during transcription may
take place through termination sites similar to those found in prokaryotes.

(2) However, the nature of individual sites is not known. Such termination sites are believed to
be present away (sometimes up to one kilobase away from the site of the 3´end of mRNA).

POST TRANSCRIPTIONAL MODIFICATIONS

Pre-mRNA Processing
In bacterial cells, transcription and translation take place simultaneously; while the 3′ end of an
mRNA is undergoing transcription, ribosomes attach to the Shine–Dalgarno sequence near the 5′
end and begin translation. Because transcription and translation are coupled, bacterial mRNA has
little opportunity to be modified before protein synthesis. In contrast, transcription and translation
are separated in both time and space in eukaryotic cells. Transcription takes place in the nucleus,
whereas translation takes place in the cytoplasm; this separation provides an opportunity for
eukaryotic RNA to be modified before it is translated. Indeed, eukaryotic mRNA is extensively
altered after transcription. Changes are made to the 5′ end, the 3′ end, and the protein coding
section of the RNA molecule.

The Addition of the 5′ Cap

One type of modification of eukaryotic pre-mRNA is the addition at its 5′ end of a structure called a
5′ cap. The cap consists of an extra nucleotide at the 5′ end of the mRNA and methyl groups (CH3)
on the base in the newly added nucleotide and on the 2′-OH group of the sugar of one or more
nucleotides at the 5′ end (Figure 14.6). The addition of the cap takes place rapidly after the
initiation of transcription and, as will be discussed in more depth in Chapter 15, the 5′ cap functions
in the initiation of translation. Cap-binding proteins recognize the cap and attach to it; a ribosome
then binds to these proteins and moves downstream along the mRNA until the start codon is
reached and translation begins. The presence of a 5′ cap also increases the stability of mRNA and
influences the removal of introns.
Three phosphate groups are present at the 5′ end of all RNA molecules because phosphate groups
are not cleaved from the first ribonucleoside triphosphate in the transcription reaction. The 5′ end of
pre-mRNA can be represented as 5′–pppNpNpN . . . , in which the letter “N” represents a
ribonucleotide and “p” represents a phosphate. Shortly after the initiation of transcription, one of
these phosphate groups is removed and a guanine nucleotide is added. This guanine nucleotide is
attached to the premRNA by a unique 5′–5′ bond, which is quite different from the usual 5′–3′
phosphodiester bond that joins all the other nucleotides in RNA. One or more methyl groups are
then added to the 5′ end; the first of these methyl groups is added
to position 7 of the base of the terminal guanine nucleotide, making the base 7-methylguanine.
Next, a methyl group may be added to the 2′ position of the sugar in the second and third
nucleotides, as shown in Figure 14.6. Rarely, additional methyl groups may be attached to the bases
of the second and third nucleotides of the pre-mRNA. Several different enzymes take part in the
addition of the 5′ cap. The initial step is carried out by an enzyme that associates with RNA
polymerase II. Because neither RNA polymerase I nor RNA polymerase III have this associated
enzyme, RNA molecules transcribed by these polymerases (rRNAs, tRNAs, and some snRNAs) are
not capped.
The Addition of the Poly(A) Tail

A second type of modification to eukaryotic mRNA is the addition of 50 to 250 or more adenine
nucleotides at the 3′ end, forming a poly(A) tail. These nucleotides are not encoded in the DNA but
are added after transcription in a process termed polyadenylation. Many eukaryotic genes
transcribed by RNA polymerase II are transcribed well beyond the end of the coding sequence;
most of the extra material at the 3′ end is then
cleaved and the poly(A) tail is added. For some pre-mRNA molecules, more than 1000 nucleotides
may be removed from the 3′ end before polyadenylation. Processing of the 3′ end of pre-mRNA
requires sequences both upstream and downstream of the cleavage site. The consensus sequence
AAUAAA is usually from 11 to 30 nucleotides upstream of the cleavage site and determines the
point at which cleavage will take place. A sequence rich in uracil nucleotides (or in guanine and
uracil nucleotides) is typically downstream of the cleavage site. A large number of proteins take
part in finding the cleavage site and removing the 3′ end. After cleavage has been completed,
adenine nucleotides are added to the new 3′ end, creating the poly(A) tail.
The poly(A) tail confers stability on many mRNAs, increasing the time during which the mRNA
remains intact
and available for translation before it is degraded by cellular enzymes. The stability conferred by
the poly(A) tail depends on the proteins that attach to the tail and on its length. The poly(A) tail also
facilitates attachment of the ribosome to the mRNA. Poly(U) tails are added to the 3′ ends of some
mRNAs, microRNAs, and small nuclear RNAs. Although the function of poly(U) tails is still under
investigation, evidence suggests that poly(U) tails on some mRNAs may facilitate their

degradation.
RNA Splicing
The other major type of modification of eukaryotic premRNA is the removal of introns by RNA
splicing. This
modification takes place in the nucleus, before the RNA moves to the cytoplasm.
Consensus sequences and the spliceosome
Splicing requires the presence of three sequences in the intron. One end of the intron is referred to
as the 5′ splice site, and the other end is the 3′ splice site (Figure 14.8); these splice sites possess
short consensus sequences. Most introns in premRNAs begin with GU and end with AG, indicating
that these sequences play a crucial role in splicing. Indeed, changing a single nucleotide at either of
these sites prevents splicing.
The third sequence important for splicing is at the branch point, which is an adenine nucleotide
that lies
from 18 to 40 nucleotides upstream of the 3′ splice site The sequence surrounding the branch point
does not have a strong consensus. The deletion or mutation of the adenine nucleotide at the branch
point prevents splicing. Splicing takes place within a large structure called the spliceosome, which
is one of the largest and most complex of all molecular complexes. The spliceosome consists of
five RNA molecules and almost 300 proteins.
The RNA components are small nuclear RNAs ranging in length from 107 to 210 nucleotides; these
snRNAs associate with proteins to form small ribonucleoprotein particles (snRNPs).

Each snRNP contains a single snRNA molecule and multiple proteins.

The spliceosome is composed of five snRNPs (U1, U2, U4, U5, and U6), and some proteins not
associated with an snRNA.
The process of splicing Before splicing takes place, an intron lies between an upstream exon (exon
1) and a
downstream exon (exon 2), PremRNA is spliced in two distinct steps. In the first step of splicing,
the pre-mRNA is cut at the 5′ splice site. This cut frees exon 1 from the intron, and the 5′ end of the
intron attaches to the branch point; that is, the intron folds back on itself, forming a structure called
a lariat. In this reaction, the guanine nucleotide in the consensus sequence at the 5′splice site bonds
with the adenine nucleotide at the branch point through a transesterification reaction. The result is
that the 5′ phosphate group of the guanine nucleotide is now attached to the 2′-OH group of the
adenine nucleotide at the branch point.
In the second step of RNA splicing, a cut is made at the 3′ splice site and, simultaneously, the 3′ end
of exon 1 becomes covalently attached (spliced) to the 5′ end of exon 2. The intron is released as a
lariat. The intron becomes linear when the bond breaks at the branch point and is then rapidly
degraded by nuclear enzymes. The mature mRNA consisting of the exons spliced together is
exported to the cytoplasm, where it is translated. These splicing reactions take place within the
spliceosome, which assembles on the pre-mRNA in a step-bystep fashion and carries out the
splicing reactions. A key feature of the process is a series of interactions between the mRNA and
the snRNAs and between different snRNAs. These interactions depend on complementary base
pairing between the different RNA molecules and bring the essential components of the pre-mRNA
transcript and the spliceosome close together, which make splicing possible.
Self-splicing introns

Some introns are self-splicing, meaning that they possess the ability to remove themselves from an
RNA molecule. These self-splicing introns fall into two major categories.

Group I introns are found in a variety of genes, including some rRNA genes in protists, some
mitochondrial genes in fungi, and even some bacteriophage genes. Although the lengths of group I
introns vary, all of them fold into a common secondary structure with nine looped stems, which are
necessary for splicing.

Group II introns, present in some mitochondrial genes, also have the ability to self-splice. All group
II introns also fold into secondary structures. The splicing of group II introns is accomplished by a
mechanism that has some similarities to the spliceosomal-mediated splicing of nuclear genes, and
splicing generates a lariat structure.

Because of these similarities, group II introns and nuclear pre-mRNA introns have been suggested to
be evolutionarily related; perhaps the nuclear introns evolved from self-splicing group II introns and
later adopted the proteins and snRNAs of the spliceosome to carry out the splicing reaction.
Alternative Processing Pathways
A finding that complicates the view of a gene as a sequence of nucleotides that specifies the amino
acid sequence of a protein (see section on The Concept of the Gene Revisited) is the existence of
alternative processing pathways. In these pathways, a single pre-mRNA is processed in different
ways to produce alternative types of mRNA, resulting in the production of different proteins from
the same DNA sequence.
One type of alternative processing is alternative splicing, in which the same pre-mRNA can be
spliced in more than one way to yield multiple mRNAs that are translated into different amino acid
sequences and thus different proteins. Another type of alternative processing requires the use of
multiple 3′ cleavage sites, where two or more potential sites for cleavage and polyadenylation are
present in the pre-mRNA. In the example, cleavage at the first site produces a relatively short
mRNA compared with the mRNA produced through cleavage at the second site. The use of an
alternative cleavage site may or may not produce a different protein, depending on whether the
position of the site is before or after the termination codon.
Both alternative splicing and multiple 3′ cleavage sites can exist in the same pre-mRNA transcript.
An example is seen in the mammalian gene that encodes calcitonin; this gene contains six exons
and five introns. The entire gene is transcribed into pre-mRNA.
There are two possible 3′ cleavage sites. In cells of the thyroid gland, 3′ cleavage and
polyadenylation take place after the fourth exon to produce a mature mRNA consisting of exons 1,
2, 3, and 4. This mRNA is translated into the hormone calcitonin.
RNA Editing

In humans, for example, a gene is transcribed into mRNA that encodes a lipid-transporting
polypeptide called apolipoprotein-B100, which has 4563 amino acids and is synthesized in liver cells.

A truncated form of the protein called apolipoprotein-B48—with only 2153 amino acids—is
synthesized in intestinal cells through editing of the apolipoprotein-B100 RNA.

In this editing, an enzyme deaminates a cytosine base, converting it into uracil. This conversion
changes a codon that specifies the amino acid glutamine into a stop codon that prematurely terminates
translation, resulting in the shortened protein.
RNA interference (RNAi) is a powerful and precise mechanism used by eukaryotic cells to limit the
invasion of foreign genes (from viruses and transposons) and to censor the expression of their own
genes.

RNA interference is triggered by double-stranded RNA molecules, which may arise in several ways:

by the transcription of inverted repeats into an RNA molecule that then base pairs with itself
to form double-stranded RNA;

by the simultaneous transcription of two different RNA molecules that are complementary to
one another and that pair, forming double-stranded RNA;

or by infection by viruses that make double-stranded RNA. T

these double-stranded RNA molecules are chopped up by an enzyme appropriately called Dicer,
resulting in tiny RNA molecules that are unwound to produce siRNAs and miRNAs (see Figure
14.23).

Some geneticists speculate that RNA interference evolved as a defense mechanism against RNA
viruses and transposable elements that move through RNA intermediates (see Chapter 11); indeed,
some have called RNAi the immune system of the genome.

However, RNA interference is also responsible for regulating a number of key genetic and
developmental processes, including changes in chromatin structure, translation, cell fate and
proliferation, and cell death.

Geneticists also use the RNAi machinery as an effective tool for blocking the expression of specific
genes

Types of Small RNAs

Two abundant classes of RNA molecules that function in RNA interference in eukaryotes are small
interfering RNAs and microRNAs.

They have a number of features in common and their functions overlap considerably. Both are about
22 nucleotides long.

Small interfering RNAs arise from the cleavage of mRNAs,RNA transposons, and RNA viruses.
Some miRNAs are cleaved from RNA molecules transcribed from sequences that encode miRNA
only, but others are encoded in the introns and exons of mRNAs.

Each miRNA is cleaved from a single-stranded RNA precursor that forms small hairpins, whereas
multiple siRNAs are produced from the cleavage of an RNA duplex consisting of two different RNA
molecules.

Usually, siRNAs have exact complementarity with their target mRNA or DNA sequences and
suppress gene expression by degrading mRNA or inhibiting transcription, whereas miRNAs often
have limited complementarity with their target mRNAs and often suppress gene expression by
inhibiting translation.

Finally, miRNAs usually silence genes that are distinct from those from which the miRNAs were
transcribed, whereas siRNAs typically silence the genes from which the siRNAs were transcribed.

Note, however, that these differences between siRNAs and miRNAs are not hard and fast, and
scientists are increasingly finding small RNAs that exhibit characteristics of both. For example, some
miRNAs have exact complementarity with mRNA sequences and cleave these sequences,
characteristics that are usually associated with siRNAs.
Processing and Function of MicroRNAs

MicroRNAs have been found in all eukaryotic organisms examined to date, as well as viruses; they
control the expression of genes taking part in many biologicalprocesses, including growth,
development, and metabolism.
Humans have more than 450 distinct miRNAs; scientists estimate that more than one-third of all
human genes are regulated by miRNAs.

Most miRNA genes are found in regions of noncoding DNA or within the introns of other genes.

The genes that encode miRNAs are transcribed into longer precursors, called primary miRNA (pri-
miRNA), that range from several hundred to several thousand nucleotides in length.

The pri-miRNA is then cleaved into one or more smaller RNA molecules with a hairpin.

Dicer (endoribonuclease Dicer or helicase with RNase motif) binds to this hairpin structure and
removes the terminal loop. One of the miRNA strands is incorporated into the RISC (RNA induced
Silencing Complex); the other strand is released and degraded.

The RISC attaches to a complementary sequence on the mRNA, usually in the 3′ untranslated region
of the mRNA.

The region of close complementarity, called the seed region, is quite short, usually only about seven
nucleotides long.

Because the seed sequence is so short, each miRNA can potentially pair with sequences on hundreds
of different mRNAs. Furthermore, a single mRNA molecule may possess multiple miRNA-binding
sites. The inhibition of translation may require binding
INHIBITORS OF TRANSCRIPTION
Rifampicin- binds with Beta subunit of prokaryotic RNA polymerase,
• It is an inhibitor of prokaryotic transcription initiation.
• It binds only to bacterial RNA polymerase but not to eukaryotic RNA polymerases.
• Therefore, Rifampicin is a powerful drug for treatment of bacterial infections.
• Used for the treatment of tuberculosis and leprosy
Actinomycin D
• Actinomycin D- Intercalates with DNA strands
• Actinomycins inhibit both DNA synthesis and RNA synthesis by blocking chain elongation.
• They interact with G·C base pairs as they require the 2-amino group of guanine for binding.
• Actinomycins are used as anticancer Drugs
Mitomycin
• Mitomycin- Intercalates with DNA strands
• blocks transcription,
• used as anticancer drug

Alpha amanitin
• Alpha amanitin is a molecule made from the “death cap” mushroom and is a known potent
inhibitor RNA polymerase.
• One single mushroom could very easily lead to a fast death in 10 days.
• The mechanism of action is that alpha amanitin inhibits RNA polymerase –II at both the initiation
and elongation states of transcription.
REFERENCES
GENETICS: A conceptual approach, 4th Edition, Benjamin A. Pierce, W. H. Freeman and company
England; 2006
Cell Biology, Genetics, Molecular Biology, Evolution and Ecology, P.S. Verma, V.K. Agarwal, S.
Chand & Company Ltd, 2005
Principles of Molecular Biology, Veer Bala Rastogi, Medtech, 2016
Molecular Biology of the Cell. 4th edition. Alberts B, Johnson A, Lewis J, et al. New York: Garland
Science; 2002.
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