Pharmaceutical Biotechnology

Dr. Rezwana Ahmed

Department of Pharmaceutical Sciences
North South University
The Central Dogma

The term gene expression refers to

the process by which the information
encoded in a DNA sequence is
translated into a product that has
some effect on a cell or organism. In
cases where the final product of the
gene is a protein, gene expression
includes both transcription and
translation. When an RNA molecule is
the gene’s final product, however,
gene expression does not require
translation.
 Transcription
Transcription is the process by which the cell copies the nucleotide
sequence of a gene into an RNA.
• Transcription begins with the opening and
unwinding of a small portion of the DNA
double helix to expose the bases on each
DNA strand.
• One of the two strands of the DNA double
helix acts as a template for the synthesis of
RNA.
• Ribonucleotides are added, one by one, to
the growing RNA chain. The nucleotide
sequence of the RNA chain is determined
by complementary base-pairing with the
DNA template. The enzyme RNA
polymerase covalently attaches the correct
incoming ribonucleotide to the growing RNA
chain.
• The RNA chain produced by transcription—the RNA transcript—is therefore
elongated one nucleotide at a time and has a nucleotide sequence exactly
complementary to the strand of DNA used as the template.
 Transcription

Figure 7–7 DNA is transcribed into RNA by the enzyme RNA polymerase

• RNA polymerase moves stepwise along the DNA, unwinding the DNA helix in
front of it. As it progresses, the polymerase adds ribonucleotides to the RNA
chain, using an exposed DNA strand as a template. The resulting RNA transcript
is thus single-stranded and complementary to this template strand.
• As the polymerase moves along the DNA template (in the 3′-to-5′ direction), it
displaces the newly formed RNA, allowing the two strands of DNA behind the
polymerase to rewind.
• A short region of hybrid DNA/RNA helix forms only transiently, causing a “window”
of DNA/RNA helix to move along the DNA with the polymerase.
 Eukaryotic Transcription

• Eukaryotic cells have three polymerases—RNA

polymerase I, RNA polymerase II, and RNA polymerase III.
These polymerases are responsible for transcribing
different types of genes and hence RNA products.

• Eukaryotic RNA polymerases require the assistance of a

large set of accessory proteins, the general transcription
factors, to help initiate transcription.

• Eukaryotic transcription initiation must take into account the

packing of DNA into nucleosomes and more compact forms
of chromatin structure.
Eukaryotic RNA Polymerase Requires General Transcription
Factors
• A promoter is part of a gene which contains a specific sequence of
nucleotides that lies immediately upstream of the starting point for RNA
synthesis. It is the location where RNA polymerase can bind.

• In eukaryotic cells, RNA polymerase cannot make RNA by itself, it requires an

assembly of accessory proteins, called general transcription factors.

• These general transcription factors assemble on the promoter, where they

position the RNA polymerase and pull apart the DNA double helix to expose
the template strand, allowing the polymerase to begin transcription.
 Eukaryotic RNA Polymerase Requires General
Transcription Factors

• The assembly process begins with the

binding of the general transcription factor
TFIID to the TATA box promoter region. The
TATA box is typically located 25 nucleotides
upstream from the transcription start site.

• TFIID binding causes a dramatic local

distortion in the DNA double helix , which
helps in the assembly of other proteins at the
promoter.

• Then the other factors assemble, along with

RNA polymerase II, to form a complete
transcription initiation complex.

Figure 7–12
 Eukaryotic RNA Polymerase Requires General
Transcription Factors

• After RNA polymerase II has been positioned on

the promoter, it must be released from the
complex of general transcription factors to start its
work. The general transcription factor TFIIH helps
to release RNA polymerase by adding phosphate
groups to the tail of RNA polymerase.

• RNA polymerase now transcribes the gene.

• When RNA polymerase finishes its work (i.e.

transcription of gene is finished), it is released
from the DNA; the phosphates on its tail are
removed, and the polymerase is then ready to find
a new promoter.
 Eukaryotic mRNAs Are Processed in the Nucleus

• A eukaryotic RNA goes through several RNA processing steps, which

include capping, splicing and polyadenylation.
• These steps take place as the RNA is being synthesized.
• The enzymes responsible for RNA processing ride on the phosphorylated
tail of eukaryotic RNA polymerase II as it synthesizes an RNA molecule,
and they process the transcript as it emerges from the polymerase.

Figure 7–15 Phosphorylation of the tail of RNA polymerase II allows RNA-processing

proteins to assemble there.
 Eukaryotic mRNAs Are Processed in the Nucleus

Capping and polyadenylation occur only on RNA transcripts destined to

become mRNA molecules (called precursor mRNAs, or pre-mRNAs).
1. RNA capping modifies the 5′ end of the RNA transcript. The RNA is
capped by the addition of an atypical nucleotide—a guanine (G)
nucleotide bearing a methyl group.
2. Polyadenylation provides a newly transcribed mRNA with a special
structure at its 3′ end. An enzyme adds a series of repeated adenine (A)
nucleotides to the 3’ end. This poly-A tail is generally a few hundred
nucleotides long.

Figure 7–16
 Eukaryotic mRNAs Are Processed in the Nucleus

The purpose of capping and polyadenylation are:

 mark the RNA molecule as an mRNA
 increase the stability of an eukaryotic mRNA
 facilitate its export from the nucleus to the cytoplasm
 help the translation machinery to recognize the mRNA
 In Eukaryotes, Protein-Coding Genes Are Interrupted by Noncoding Sequences
Called Introns

• In bacteria, the coding DNA sequences are uninterrupted, this DNA is transcribed
into an mRNA. The mRNA can be translated into protein without any further
processing.

• In eukaryotes, most protein-coding genes, have their coding sequences

interrupted by long, noncoding, intervening sequences called introns. The
scattered pieces of coding sequence—called expressed sequences or exons—
are usually shorter than the introns, and they often represent only a small fraction
of the total length of the gene.
 Introns Are Removed From Pre-mRNAs by RNA Splicing

pre-mRNA

• When a eukaryotic mRNA is first produced, it contains both the introns and
exons. The non-coding sequences- introns are removed by a process
called splicing.
• After capping, and as RNA polymerase II continues to transcribe the gene,
the process of RNA splicing begins, in which the introns are removed from
the newly synthesized RNA and the exons are stitched together.
• The RNA splicing is performed by some RNA molecules with additional
proteins. This complex of RNA+ proteins is called spliceosome.
 Purpose of Alternative splicing

Figure 7–22 Some pre-mRNAs undergo alternative RNA splicing to produce various mRNAs
and proteins from the same gene.

• The intron–exon type of gene arrangement allows the transcripts of

many eukaryotic genes to be spliced in different ways, each of which
can produce a distinct protein.
• Such alternative splicing allows many different proteins to be produced
from the same gene.
• Thus RNA splicing enables eukaryotes to increase the coding potential
of their genomes.
 Mature Eukaryotic mRNAs Are Exported from the Nucleus

• Once a transcript has been spliced and its cap and poly-A tail has been
added, the RNA is now a functional mRNA called mature mRNA. It can
leave the nucleus so that translation can occur.
• Before a mature mRNA is exported to the cytoplasm, it must be bound to
an appropriate set of proteins. These proteins include poly-A–binding
proteins, a cap-binding complex, and proteins that bind to mRNAs that
have been appropriately spliced.
• The mRNA then moves into the cytoplasm through the nuclear pore
complexes, which control which macromolecules can enter or leave the
nucleus.
 Difference between eukaryotic and prokaryotic transcription
initiation

• Bacteria contain a single type of RNA polymerase, eukaryotic cells

have three—RNA polymerase I, RNA polymerase II, and RNA
polymerase III. These polymerases are responsible for transcribing
different types of genes.

• The bacterial RNA polymerase is able to initiate transcription on its

own, it does not need the general transcription factors. Eukaryotic RNA
polymerases require the assistance of the general transcription factors
to initiate transcription.

• Eukaryotic transcription initiation must take into account the packing of

DNA into nucleosomes and more compact forms of chromatin structure,
whereas the prokaryotic cells do not have such complex structures of
DNA.
 Difference between eukaryotic and prokaryotic transcription

Figure 7–24 Prokaryotes and eukaryotes handle their RNA transcripts differently
 Difference between eukaryotic and prokaryotic transcription

• In eukaryotic cells, the pre-mRNA molecule produced by transcription

contains both intron and exon sequences. Its two ends are modified, and
the introns are removed by RNA splicing. The resulting mRNA is then
transported from the nucleus to the cytoplasm, where it is translated into
protein. In prokaryotes, the production of mRNA molecules is simpler.
The 5′ end of an mRNA molecule is produced by the initiation of
transcription by RNA polymerase, and the 3′ end is produced by the
termination of transcription, i.e. no modifications are needed. Because
prokaryotic cells lack a nucleus, transcription and translation take place
in a common compartment.
Figure 7–2 A cell can express different genes at different rates.