Assignment

On,

How the genes are regulated & expressed?

Course: Introduction to Molecular Biology

Course code: MIC206

Submitted to: Mahbubul Hassan Siddiqee

Lecturer, MNS Department

Submitted by:

15226004- Sumaiya Sultana

16126009- Farhana Khanam

16126003- Fatema Tuj Johora Shumona

Submission date: 3 April, 2017

Some questions may arise…..

1. What is gene regulation?

2. How genes are controlled?
3. Why transcription is so important?
4. Why does RNA have the base uracil instead of thymine?
5. How RNA synthesis occurs without any primer?
6. What are promoters?
7. How does bacterial RNA polymerase locate the promoter?
8. What is the benefit of the coding strand if it doesn't get transcribed and only the template
strand gets transcribed?
9. Which factors are needed for transcription?
10. Why does eukaryotic transcription require several RNA polymerases unlike prokaryotic
transcription?
11. Why pre-mRNA is modified during eukaryotic transcription?
12. Why can transcription and translation happen simultaneously for an mRNA in bacteria?
13. Why the introns are there when they are to be removed? What are the functions of
introns?
14. Does the presence of introns indicate something related to evolution?
15. What will happen if transcription does not occur properly and an error remains during
synthesis?
16. Why doesn’t RNA polymerase need to proofread?
17.
Outline of the assignment…….

 Transfer of Genetic Information: The Central Dogma

 The Process of Gene Expression
 Transcription in Prokaryotes
 RNA polymerases: Complex enzymes
 Initiation, elongation, termination
 Transcription in Eukaryotes
 Initiation, elongation, termination
 The five Eukaryotic RNA Polymerases
 Structure of RNA polymerase II
 Transcription factors
 Modifications of pre-mRNA in eukaryotes
 5’ cap
 3’ ploy(A) tail
 RNA splicing
 Types of RNA molecule
 Introns: Biological significance
 Summary
The central dogma of biology is that information stored in DNA is transferred to RNA
molecules during transcription and to proteins during translation.

According to the central dogma of molecular biology, genetic information usually flows:

(1) From DNA to DNA during its transmission from generation to generation

(2) From DNA to protein during its phenotypic expression in an organism

The transfer of genetic information from DNA to protein involves two steps:
 Transcription, the transfer of the genetic information from DNA to RNA
 Translation, the transfer of information from RNA to protein

Each cell expresses, or turns on, only a fraction of its genes. The rest of the genes are
repressed, or turned off. The process of turning genes on and off is known as gene regulation.
Gene regulation can occur at any point during gene expression, but most commonly occurs at
the level of transcription (when the information in a gene’s DNA is transferred to mRNA).
During transcription the base thymine is replaced with uracil. As in RNA, Uracil is present
instead of thymine because RNA remains in the cell for a very short period of time. Uracil is
energetically less expensive to produce then thymine. And that is why Uracil is present in
RNA instead of thymine.

Signals from the environment or from other cells activate proteins called transcription factors.
These proteins bind to regulatory regions of a gene and increase or decrease the level of
transcription. By controlling the level of transcription, this process can determine the amount
of protein product that is made by a gene at any given time.

As our bodies cannot use the food as it is when it enters the digestive system. The process of
chemical digestion uses various kinds of proteins and enzymes to break down the food into a
useable nutrient. The instructions to make proteins are contained in DNA. Transcription is
important because the information that is contained in DNA cannot be transferred to other
cells in the body because DNA can't leave the nucleus. This means that the information is
carried over to the RNA cell which can transfer the information outside of the nucleus. The
mRNA is formed through the process of transcription. From this, the importance of
transcription can easily be understood.

Transcription
The principal enzyme responsible for RNA synthesis is RNA polymerase. The synthesis of
RNA is similar to that of DNA, and like DNA polymerase, RNA polymerase catalyzes the
growth of RNA chains always in the 5′ to 3′ direction. Unlike DNA polymerase, however,
RNA polymerase does not require a preformed primer to initiate the synthesis of RNA.
Instead, transcription initiates de novo at specific sites at the beginning of genes. The
initiation process is particularly important because this is the primary step at which
transcription is regulated.

Promoter recognition

A promoter is a region of DNA that initiates transcription of a particular gene. RNA

polymerase starts transcription by recognizing the promoter. The promoter isn't copied into
RNA, but it is, nonetheless, an important piece of genetic information. The information in a
promoter was determined by lining up a large number of promoters and counting how many
times a particular base appeared at a given position in the various promoter sequences. A
couple of important points exist about the consensus. First, not all bases in the consensus are
conserved to the same amount. The bases marked with bold type and underlined are more
conserved than the others, and the ‐10 region is more conserved overall than is the ‐35 region.
Secondly, the promoter sequence is asymmetrical; that is, it reads differently in one direction
than in the other.
Transcription in Prokaryotes
E. coli RNA polymerase is a complex enzyme made up of multiple polypeptide chains. The
intact enzyme consists of four different types of polypeptides. The complete RNA
polymerase molecule, the holoenzyme, has the composition α2ββ′σ. The subunits are
involved in the assembly of the tetrameric core (α2ββ′) of RNA polymerase. The subunit
contains the ribonucleoside triphosphate binding site, and the subunit harbors the DNA
template-binding region. One subunit, the sigma factor, is involved only in the initiation of
transcription; it plays no role in chain elongation. After RNA chain initiation has occurred,
the factor is released, and chain elongation is catalyzed by the core enzyme. The function of
sigma is to recognize and bind RNA polymerase to the transcription initiation or promoter
sites in DNA. The core enzyme will catalyze RNA synthesis from DNA templates in vitro
but, in so doing, it will initiate RNA chains at random sites on both strands of DNA. In
contrast, the holoenzyme (α 2ββ′) initiates RNA chains in vitro only at sites used in vivo.

Initiation of RNA chains

RNA polymerase only goes one direction from a promoter and only one strand of DNA is
used as a template at any one time because if both strands are used to make mRNA, it can't
optimize one without messing up the other, and vice versa. Moreover, produced mRNA must
be single stranded to be fit in the ribosome. To provide this template strand, the initiation of
transcription involves a short unwinding of the DNA double helix. This is accomplished in a
two‐step fashion. First, RNA polymerase binds to the promoter to form the closed complex,
which is relatively weak. Then, the double‐stranded DNA goes through a conformational
change to form the much stronger open complex through opening of the base pairs at the ‐10
sequence. The initiator nucleotide binds to the complex and the first phosphodiester bonds are
made, accompanied by release of δ. The remaining core polymerase is now in the elongation
mode. Several experimental observations support the picture presented in the next figure,
namely the fact that less than one δ exists in the cell per core enzyme in each cell.
Elongation of RNA chains

Elongation is the function of the RNA polymerase core enzyme. RNA polymerase moves
along the template, locally “unzipping” the DNA double helix. This allows a transient base
pairing between the incoming nucleotide and newly‐synthesized RNA and the DNA template
strand. As it is made, the RNA transcript forms secondary structure through intra‐strand base
pairing. The average speed of transcription is about 40 nucleotides per second, much slower
than DNA polymerase. Other protein factors may bind to polymerase and alter the rate of
transcription and some specific sequences are transcribed more slowly than others are.
Eventually, RNA polymerase must come to the end of the region to be transcribed.

Termination of RNA chains

There are two major termination strategies found in bacteria: Rho-dependent and Rho-
independent.
In Rho-dependent termination, the RNA contains a binding site for a protein called Rho
factor. Rho factor binds to this sequence and starts "climbing" up the transcript towards RNA
polymerase.
When it catches up with the polymerase at the transcription bubble, Rho pulls the RNA
transcript and the template DNA strand apart, releasing the RNA molecule and ending
transcription. Another sequence found later in the DNA, called the transcription stop point,
causes RNA polymerase to pause and thus helps Rho catch up.4^44start superscript, 4, end
superscript
Rho-independent termination depends on specific sequences in the DNA template strand.
As the RNA polymerase approaches the end of the gene being transcribed, it hits a region
rich in C and G nucleotides. The RNA transcribed from this region folds back on itself, and
the complementary C and G nucleotides bind together. The result is a stable hairpin that
causes the polymerase to stall.

In a terminator, the hairpin is followed by stretches of U nucleotides in the RNA, which

match up with A nucleotides in the template DNA. The complementary U-A region of the
RNA transcript forms only a weak interaction with the template DNA. This, coupled with the
stalled polymerase, produces enough instability for the enzyme to fall off and liberate the
new RNA transcript.
Transcription in Eukaryotes
Prokaryotes and eukaryotes perform fundamentally the same process of transcription, with a
few key differences. The most important difference between prokaryotes and eukaryotes is
the latter’s membrane-bound nucleus and organelles. With the genes bound in a nucleus, the
eukaryotic cell must be able to transport its mRNA to the cytoplasm and must protect its
mRNA from degrading before it is translated.

Initiation of RNA chains

Unlike the prokaryotic polymerase that can bind to a DNA template on its own, eukaryotes
require several other proteins, called transcription factors, to first bind to the promoter region
and then help recruit the appropriate polymerase.

The five Eukaryotic RNA Polymerases

The features of eukaryotic mRNA synthesis are markedly more complex those of
prokaryotes. In eukaryotes there is five RNA polymerase whereas in prokaryotes there is only
one RNA polymerase. In eukaryotes the transcription process needs to be accurate to be
functional and it is also a lengthy process in comparison to prokaryotes. The prokaryotes
have short life-span so it is a waste of time for them to produce five polymerase enzymes.

Table 15.3.1: Locations, Products, and Sensitivities of the Three Eukaryotic RNA
Polymerases
RNA polymerase II is located in the nucleus and synthesizes all protein-coding nuclear pre-
mRNAs. Eukaryotic pre-mRNAs undergo extensive processing after transcription but before
translation. For clarity, this module’s discussion of transcription and translation in eukaryotes
will use the term “mRNAs” to describe only the mature, processed molecules that are ready
to be translated. RNA polymerase II is responsible for transcribing the overwhelming
majority of eukaryotic genes.

RNA polymerase III is also located in the nucleus. This polymerase transcribes a variety of
structural RNAs that includes the 5S pre-rRNA, transfer pre-RNAs (pre-tRNAs), and small
nuclear pre-RNAs. The tRNAs have a critical role in translation; they serve as the adaptor
molecules between the mRNA template and the growing polypeptide chain. Small nuclear
RNAs have a variety of functions, including “splicing” pre-mRNAs and regulating
transcription factors.

Structure of an RNA polymerase II Promoter

Eukaryotic promoters are much larger and more complex than prokaryotic promoters, but
both have a TATA box. For example, in the mouse thymidine kinase gene, the TATA box is
located at approximately -30 relative to the initiation (+1) site (Figure 15.3.1). For this gene,
the exact TATA box sequence is TATAAAA, as read in the 5' to 3' direction on the
nontemplate strand. This sequence is not identical to the E. coli TATA box, but it conserves
the A–T rich element. The thermo stability of A–T bonds is low and this helps the DNA
template to locally unwind in preparation for transcription.
Figure 15.3.1: A generalized promoter of a gene transcribed by RNA polymerase II is
shown. Transcription factors recognize the promoter. RNA polymerase II then binds and
forms the transcription initiation complex.

Transcription Factors for RNA polymerase II

Factor Protected Fragment

TFIID -42 to -17 (binds TATA box)
TFIIA -80 to -17
TFIIB -80 to -17 and -10 to +10
RNA Polymerase II -80 to +15
TFIIE -80 to +30

Promoter Structures for RNA Polymerases I and III

In eukaryotes, the conserved promoter elements differ for genes transcribed by RNA
polymerases I, II, and III. RNA polymerase I transcribes genes that have two GC-rich
promoter sequences in the -45 to +20 regions. These sequences alone are sufficient for
transcription initiation to occur, but promoters with additional sequences in the region from
-180 to -105 upstream of the initiation site will further enhance initiation. Genes that are
transcribed by RNA polymerase III have upstream promoters or promoters that occur within
the genes themselves.

Enhancers and silencers

A typical protein-coding gene is likely to contain several enhancers which act at a distance.
These elements are usually 700–1000 bp or more away from the start of transcription. The
hallmark of enhancers is that, unlike promoter elements, they can be downstream, upstream,
or within an intron, and can function in either orientation relative to the promoter. A typical
enhancer is around 500 bp in length and contains in the order of 10 binding sites for several
different transcription factors. Each enhancer is responsible for a subset of the total gene
expression pattern. Enhancers increase gene promoter activity either in all tissues or in a
regulated manner. Similar elements that repress gene activity are called silencers.

Elongation and Termination of RNA chains

Following the formation of the preinitiation complex, the polymerase is released from the
other transcription factors, and elongation is allowed to proceed as it does in prokaryotes with
the polymerase synthesizing pre-mRNA in the 5' to 3' direction. As discussed previously,
RNA polymerase II transcribes the major share of eukaryotic genes, so this section will focus
on how this polymerase accomplishes elongation and termination.

Although the enzymatic process of elongation is essentially the same in eukaryotes and
prokaryotes, the DNA template is more complex. When eukaryotic cells are not dividing,
their genes exist as a diffuse mass of DNA and proteins called chromatin. The DNA is tightly
packaged around charged histone proteins at repeated intervals. These DNA–histone
complexes, collectively called nucleosomes, are regularly spaced and include 146 nucleotides
of DNA wound around eight histones like thread around a spool.

For polynucleotide synthesis to occur, the transcription machinery needs to move histones out
of the way every time it encounters a nucleosome. This is accomplished by a special protein
complex called FACT, which stands for “facilitates chromatin transcription.” This complex
pulls histones away from the DNA template as the polymerase moves along it. Once the pre-
mRNA is synthesized, the FACT complex replaces the histones to recreate the nucleosomes.

The termination of transcription is different for the different polymerases. Unlike in

prokaryotes, elongation by RNA polymerase II in eukaryotes takes place 1,000–2,000
nucleotides beyond the end of the gene being transcribed. This pre-mRNA tail is
subsequently removed by cleavage during mRNA processing. On the other hand, RNA
polymerases I and III require termination signals. Genes transcribed by RNA polymerase I
contain a specific 18-nucleotide sequence that is recognized by a termination protein. The
process of termination in RNA polymerase III involves an mRNA hairpin similar to rho-
independent termination of transcription in prokaryotes.

Modification of Eukaryotic pre-mRNAs

After termination, transcription is finished. An RNA transcript that is ready to be used in

translation is called a messenger RNA (mRNA).
In bacteria, RNA transcripts are ready to be translated right after transcription. In fact, they're
actually ready a little sooner than that: translation may start while transcription is still going
on!
In the diagram below, mRNAs are being transcribed from several different genes. Although
transcription is still in progress, ribosomes have attached each mRNA and begun to translate
it into protein. When an mRNA is being translated by multiple ribosomes, the mRNA and
ribosomes together are said to form a polyribosome.
Transcription and translation can happen simultaneously for an mRNA in bacteria as these
processes occur in the same 5' to 3' direction. That means one can follow or "chase" another
that's still occurring. Also, in bacteria, there are no internal membrane compartments to
separate transcription from translation. But in eukaryotes, RNA molecules need to go through
special processing steps before translation. That means translation can't start until
transcription and RNA processing are fully finished. Moreover, there are internal membrane
compartments to separate transcription from translation.

Eukaryotic gene transcripts usually undergo three major modifications:

1. Addition of 7-methyl guanosine caps to 5’ termini:

Early in the elongation process, the 5’ ends of eukaryotic pre-mRNAs are modified by
addition of 7-methyl guanosine caps. These caps are added when the growing RNA chains
are only about 30 nucleotides long. The 7-MG cap contains an unusual 5’-5’ triphosphate
linkage and two or more methyl groups. These caps are added co-transcriptionally by the
biosynthetic pathway. The 7-MG caps are recognized by protein factors involved in the
initiation of translation and also help protect the growing RNA chains from degradation by
nucleases.

2. Addition of poly(A) tails to 3’ ends:

A poly(A) tail (a 20-200 nucleotide polyadenosine tract, A’s) is added to the 3’ end of the
transcript. The 3’ end is generated by cleavage rather than by termination. The poly(A) tails
of eukaryotic mRNAs enhance their stability and play an important role in their transport
from the nucleus to the cytoplasm.
3. RNA splicing:

The third big RNA processing event is RNA splicing. In RNA splicing, specific parts of the
pre-mRNA, called introns are recognized and removed by a protein and RNA complex,
called the spliceosome. Introns can be viewed as "junk" sequences that must be cut out so
the "good parts version" of the RNA molecule can be assembled.

The pieces of the RNA that are not chopped out are called exons. The exons are pasted
together by the spliceosome to make the final, mature mRNA that is shipped out of the
nucleus. It's only the exons of a gene that encode a protein. Not only do the introns not carry
information to build a protein, they actually have to be removed in order for the mRNA to
encode a protein with the right sequence. If the spliceosome fails to remove an intron, an
mRNA with extra "junk" in it will be made, and a wrong protein is will get produced during
translation.

Alternative splicing

Splicing does allow for a process called alternative splicing, in which more than one mRNA
can be made from the same gene. Through alternative splicing, eukaryotes can sneakily
encode more different proteins than the genes present in DNA.
In alternative splicing, one pre-mRNA may be spliced in either of two (or sometimes many
more than two) different ways. For example, in the diagram below, the same pre-mRNA can
be spliced in three different ways, depending on which exons are kept. This results in three
different mature mRNAs, each of which translates into a protein with a different structure.

Types of RNA molecule

 Messenger RNAs (mRNAs) — intermediates that carry genetic information from DNA
to the ribosomes.
 Transfer RNAs (tRNAs) — adaptors between amino acids and the codons in mRNA.
 Ribosomal RNAs (rRNAs) — structural and catalytic components of ribosomes.
 Small nuclear RNAs (snRNAs) — structural components of spliceosomes
 Micro RNAs (miRNAs) — short single-stranded RNAs (20 to 22 bp) that block
expression of complementary mRNAs.
 RNAi is similar to miRNA (RNA interference, double strand RNA, plant), siRNA (small
interference)
 piRNA (Piwi-interacting RNA) is a small non-coding RNA molecules

Introns: Biological significance

At present, scientists know relatively little about the biological significance of the exon–
intron structure of eukaryotic genes. Introns are highly variable in size, ranging from about 50
nucleotide pairs to thousands of nucleotide pairs in length. This fact has led to speculation
that introns may play a role in regulating gene expression. Although it is unclear how introns
regulate gene expression, new research has shown that some introns contain sequences that
can regulate gene expression in either a positive or negative fashion. Other introns contain
alternative tissue-specific promoters; still others contain sequences that enhance the
accumulation of transcripts.
The fact that introns accumulate new mutations much more rapidly than exons indicates that
many of the specific nucleotide-pair sequences of introns, excluding the ends, are not very
important.
In some cases, the different exons of genes encode different functional domains of the protein
gene products. In the case of the mammalian globin genes, the middle exon encodes the
heme-binding domain of the protein. There has been considerable speculation that the exon–
intron structure of eukaryotic genes has resulted from the evolution of new genes by the
fusion of uninterrupted (single exon) ancestral genes. If this hypothesis is correct, introns
may merely be relics of the evolutionary process.

Proofread activity of RNA polymerase

During replication the DNA polymerase has the proof read activity by which it can remove
the wrong paired nucleotide whereas RNA polymerase doesn’t have this activity. It is
assumed that RNA is working copies and it can tolerate some errors. However, new
functional and structural studies have suggested how RNAPs select the correct nucleoside
triphosphate (NTP) substrate to prevent transcription errors, and how the enzymes detect and
remove a misincorporated nucleotide during proofreading. Proofreading begins with fraying
of the misincorporated nucleotide away from the DNA template, which pauses transcription.
Subsequent backtracking of RNAP by one position enables nucleolytic cleavage of an RNA
dinucleotide that contains the misincorporated nucleotide. Since cleavage occurs at the same
active site that is used for polymerization, the RNAP proofreading mechanism differs from
that used by DNAPs, which contain a distinct nuclease specific active site.
To summarize,

 The central dogma of molecular biology is that genetic information flows from DNA
to DNA during chromosome replication, from DNA to RNA during transcription, and
from RNA to protein during translation.
 Transcription involves the synthesis of an RNA transcript complementary to one
strand of DNA of a gene.
 RNA synthesis occurs in three stages: (1) initiation, (2) elongation, and (3)
termination. KEY POINTS
 The covalent extension of RNA chains occurs within locally unwound segments of
DNA.
 Chain elongation stops when RNA polymerase encounters a transcription–termination
signal.
 Transcription, translation, and degradation of mRNA molecules often occur
simultaneously in prokaryotes.
 In eukaryotes, genes are present in the nucleus, whereas polypeptides are synthesized
in the cytoplasm.
 Messenger RNA molecules function as intermediaries that carry genetic information
from DNA to the ribosomes, where proteins are synthesized.
 RNA synthesis, catalyzed by RNA polymerases, is similar to DNA synthesis in many
respects.
 Three to five different RNA polymerases are present in eukaryotes, and each
polymerase transcribes a distinct set of genes.
 Eukaryotic gene transcripts usually undergo three major modifications: (1) the
addition of 7-methyl guanosine caps to 5’ termini, (2) the addition of poly(A) tails to
3’ ends, and (3) the excision of noncoding intron sequences.
 Introns contain some sequences that can regulate gene expression in either a positive
or negative fashion. Other introns contain alternative tissue-specific promoters; still
others contain sequences that enhance the accumulation of transcripts.