GENE STRUCTURE AND GENE EXPRESSION

Chemical structure of genes

 Genes are composed of deoxyribonucleic acid (DNA), except in some viruses, which have genes
consisting of a closely related compound called ribonucleic acid (RNA).
 A DNA molecule is composed of two chains of nucleotides that wind about each other to
resemble a twisted ladder.
 The sides of the ladder are made up of sugars and phosphates, and the rungs are formed by
bonded pairs of nitrogenous bases.
 These bases are adenine (A), guanine (G), cytosine (C), and thymine (T).
 An A on one chain bonds to a T on the other (thus forming an A–T ladder rung); similarly, a C on
one chain bonds to a G on the other.
 If the bonds between the bases are broken, the two chains unwind, and free nucleotides within
the cell attach themselves to the exposed bases of the now-separated chains.
 The free nucleotides line up along each chain according to the base-pairing rule A bonds to T, C
bonds to G.
 This process results in the creation of two identical DNA molecules from one original and is the
method by which hereditary information is passed from one generation of cells to the next.
 The sequence of bases along a strand of DNA determines the genetic code.

The central dogma theory

 The central dogma of molecular biology is a theory stating that genetic information flows only in
one direction, from DNA, to RNA, to protein, or RNA directly to protein
 It illustrates the flow of genetic information in cells
 Involves the DNA Replication (DNA creating identical molecules), Transcription (DNA
coding for the RNA) and Translation (RNA codes for the proteins).

A. Gene replication
 Replication involves the production of identical helices of DNA from one double-stranded
molecule of DNA.
Initiation Stage
 DNA replication begins when an enzyme called helicase unwinds, and unzips the DNA
molecule.
 Helicase breaks apart the hydrogen bonds holding together complementary nitrogenous
bases.
 Topoisomerase or DNA Gyrase - unwinds and rewinds DNA strands to prevent the DNA
from becoming tangled or supercoiled.
 This causes the two strands of DNA to separate into a Y shape known as the replication
fork. This area will be the template for replication to begin.
 Small molecules called single-stranded binding proteins (SSB) attach to the loose strands
of DNA to keep them from re-forming the hydrogen bonds that helicase just broke apart.
 SSB exposes the nitrogenous bases from the inside of the DNA molecule
 The creation of a new, complementary strand can begin.

Primer Binding
 DNA polymerase creates the new strand, but it needs some help in finding the correct place
to begin
 Primase lays down a short section of RNA primer
 DNA polymerase can bind to the DNA molecule and start connecting nucleotides in the
correct order to match the sequence of nitrogenous bases on the template (original) strand.
 Two strands of DNA run antiparallel to one another. This means that in the sugar-phosphate
backbone, one strand of the DNA has the sugar oriented in the “up” position, and the other
strand has the phosphate oriented in the “up” position.
 The replication fork is bi-directional; one strand is oriented in the 3' to 5' direction (leading
strand) while the other is oriented 5' to 3' (lagging strand).
 DNA polymerase is an enzyme which can only work in one direction on the DNA molecule.
 This means that one strand of DNA can be replicated in one long string, as DNA polymerase
follows helicase as it unzips the DNA molecule (leading strand).
 The other strand, however, can only be replicated in small chunks since the DNA polymerase
replicates in the opposite direction that helicase is unzipping (lagging strand).
 These small chunks of replicated DNA on the lagging strand are called Okazaki fragments.
Termination
 Once both the continuous and discontinuous strands are formed, an enzyme
called exonuclease removes all RNA primers from the original strands.
 These primers are then replaced with appropriate bases. Another exonuclease “proofreads”
the newly formed DNA to check, remove and replace any errors.
 Once DNA polymerase has replicated the DNA, another enzyme called ligase completes the
final stage of DNA replication, which is repairing the sugar-phosphate backbone.
 The ends of the parent strands consist of repeated DNA sequences called telomeres.
Telomeres act as protective caps at the end of chromosomes to prevent nearby chromosomes
from fusing.
 A special type of DNA polymerase enzyme called telomerase catalyzes the synthesis of
telomere sequences at the ends of the DNA.
 Ligase connects the gaps in the backbone between Okazaki fragments.
 Once this is complete, the DNA coils back into its classic double helix structure.
 When DNA replication is complete, there are two identical sets of double stranded DNA,
each with one strand from the original, template, DNA molecule, and one strand that was
newly synthesized during the DNA replication process.
 Because each new set of DNA contains one old and one new strand, DNA is described as
being semi-conservative.
 The new DNA strands are formed, with one strand of the parent DNA and the other is
newly synthesized, this process is called semiconservative DNA replication.
B. Gene transcription
 Transcription is the process by which the information is transferred from one strand of
the DNA to RNA by the enzyme RNA polymerase.
 The DNA strand which undergoes this process consists of three parts namely promoter,
structural gene, and a terminator.
 The DNA strand that synthesizes the RNA is called the template strand and the other
strand is called the coding strand.
Initiation
 The DNA-dependent RNA polymerase binds to the promoter and catalyzes the
polymerization in the 3′ to 5′ direction.
 RNA polymerase, which attaches to and moves along the DNA molecule until it
recognises a promoter sequence.
 Transcription factors are proteins that control the rate of transcription also bind to the
promoter sequences with RNA polymerase.
 RNA polymerase unwinds a portion of the DNA double helix, exposing the bases on
each of the two DNA strands.
Elongation
 One DNA strand (the template strand) is read in a 3′ to 5′ (three-prime to five-prime)
direction, and so provides the template for the new mRNA molecule.
 The other DNA strand is referred to as the coding strand.
 This is because its base sequence is identical to the synthesised mRNA, except for the
replacement of thiamine bases with uracil.
 RNA polymerase uses incoming ribonucleotides (the free nucleotides in the cell) to
form the new mRNA strand.
 It does this by catalysing the formation of phosphodiester bonds between adjacent
ribonucleotides, using complementary base pairing (A to U, T to A, C to G, and G to C).
 Bases can only be added to the 3′ end, so the strand elongates in a 5’ to 3’ direction.
 The newly released RNA strand further undergoes post-transcriptional modifications.
 This single chain of RNA, called messenger RNA (mRNA), then passes to the organelles
called ribosomes in RER, where the process of translation, or protein synthesis, takes
place.
 As it approaches the terminator sequence, it terminates and releases the newly
synthesized RNA strand.

Termination
 Elongation continues until the RNA polymerase encounters a stop sequence.
 At this point, transcription stops, and the RNA polymerase releases the DNA template.
Pre-translational mRNA processing (Post- transcriptional modifications)

 The mRNA which has been transcribed up to this point is referred to as pre-mRNA. Processing
must occur to convert this into mature mRNA. This includes:
1) 5′ Capping
 Capping describes the addition of a methylated guanine cap to the 5′ end of mRNA.
 Its presence is vital for the recognition of the molecule by ribosomes, and to protect the
immature molecule from degradation by RNAases.
2) Polyadenylation
 Polyadenylation describes the addition of a poly(A) tail to the 3′ end of mRNA.
 The poly(A) tail consists of multiple molecules of adenosine monophosphate.
 This helps to stabilise RNA, which is necessary as RNA is much more unstable than
DNA.
3) Splicing
 Splicing allows the genetic sequence of a single pre-mRNA to code for many different
proteins, conserving genetic material.
 This process is sequence-dependent and occurs within the transcript.
 It involves:
a) Removal of introns (non-coding sequences) via spliceosome excision
b) Joining together of exons (coding sequence) by ligation
 By the end of transcription, mature mRNA has been made. This acts as the messaging
system to allow translation and protein synthesis to occur.
 Within the mature mRNA, is the open reading frame (ORF).
 This region will be translated into protein.
 It is translated in blocks of three nucleotides, called codons.
 At the 5’ and 3’ ends, there are also untranslated regions (UTRs).
 These are not translated during protein synthesis.
C. Gene translation
 During translation, a second type of RNA, transfer RNA (tRNA), matches up the
nucleotides on mRNA with specific amino acids.
 Translation is the process by which the RNA codes for specific proteins.
 It is an active process which requires energy. This energy is provided by the charged
tRNA molecules.
 Ribosomes initiate the translation process.
 The ribosomes consist of a larger subunit and a smaller subunit.
 Larger subunit consists of two tRNA molecules placed close enough so that peptide
bond can be formed at the expense of enough energy.
 The mRNA enters the smaller subunit which is then held by the tRNA molecules of the
complementary codon present in the larger subunit.
 Two codons are held by two tRNA molecules placed close to each other and a peptide
bond is formed between them.
 As this process repeats, long polypeptide chains of amino acids are synthesized.
 Each set of three nucleotides codes for one amino acid.
 The series of amino acids built according to the sequence of nucleotides forms
a polypeptide chain
 All proteins are made from one or more linked polypeptide chains.
 Genetic code contains the information of the protein manufactured from RNA.
 There are basically three nucleotides and four nitrogenous bases, which collectively form
a triplet codon that codes for one amino acid.
 Therefore, the number of possible amino acids ranges to 4 x 4 x 4 = 64 amino acids.
 Each codon codes for only one specific amino acid and the codes are universal
irrespective of the type of organism.
 There are 20 naturally existing amino acids.
 The genetic code degenerates. This was explained by the features of the genetic code,
according to which a few amino acids are coded by more than one codon thus causing
them to degenerate.
 Out of the 64 codons, 3 are stop codons which stop the process of transcription and one
of the codons is an initiator codon i.e. AUG coding for Methionine.
Twenty amino acids
1) Alanine - ala - A
2) Arginine - arg - R
3) Asparagine - asn - N
4) Aspartic acid - asp - D
5) Cysteine - cys - C
6) Glutamine - gln - Q
7) Glutamic acid - glu - E
8) Glycine - gly - G
9) Histidine - his - H
10) Isoleucine - ile - I
11) Leucine - leu - L
12) Lysine - lys - K
13) Methionine - met - M
14) Phenylalanine - phe - F
15) Proline - pro - P
16) Serine - ser - S
17) Threonine - thr - T
18) Tryptophan - trp - W
19) Tyrosine - tyr - Y
20) Valine - val - V
Gene expression regulation

 Many of the genes within the cells of organisms are inactive much or even all of the time.
 A gene can be switched on or off, at any time, in both eukaryotes and prokaryotes.
 The regulation of genes expression differs in eukaryotes and prokaryotes.

A Gene regulation in Prokaryotes (The operon model)

 The process by which genes are activated and deactivated in bacteria is well characterized.
 Bacteria have three types of genes: structural, operator, and regulator.
 Structural genes code for the synthesis of specific polypeptides.
 Operator genes contain the code necessary to begin the process of transcribing the DNA
message of one or more structural genes into mRNA.
 Thus, structural genes are linked to an operator gene in a functional unit called an operon.
 An operon is a functioning unit of DNA containing a cluster of genes under the control of a
single promoter
 Operons include tryptophan (Trp) operon is inhibited by a chemical (tryptophan), L-arabinose
operon, Lac operon and Gal operon
 Ultimately, the activity of the operon is controlled by a regulator gene, which produces a
small protein molecule called a repressor.
 The repressor binds to the operator gene and prevents it from initiating the synthesis of the
protein(s) called for by the operon.
 The presence or absence of certain repressor molecules determines whether the operon is off or
on.

B Gene regulation in Eukaryotes

 The genes of eukaryotes, which do not have operons, are regulated independently.
 The series of events associated with gene expression in higher organisms involves multiple levels
of regulation and is often influenced by the presence or absence of molecules called transcription
factors.
 These factors influence the fundamental level of gene control, which is the rate of transcription,
and may function as activators or enhancers.
 Specific transcription factors regulate the production of RNA from genes at certain times and in
certain types of cells.
 Transcription factors bind to the promoter, or regulatory region, found in the genes of higher
organisms.
 After transcription, introns (noncoding nucleotide sequences) are excised from the primary
transcript through processes known as editing and splicing.
 The result of these processes is a functional strand of mRNA and this is a routine step in the
production of mRNA for most genes
 However, in some genes there are multiple ways to splice the primary transcript, resulting in
different mRNAs, which in turn result in different proteins.
  Some genes also are controlled at the translational and posttranslational levels.

THE LAC OPERON

 The lac operon is one of the most studied operons in E. coli.
 The lac operon is an operon, or group of genes with a single promoter (transcribed as a single
mRNA).
 The lac operon contains three genes: lacZ, lacY, and lacA.
 These genes are transcribed as a single mRNA, under control of one promoter and operator
 The genes in the operon encode proteins that allow the bacteria to use lactose as an energy
source.
 E. coli bacteria can break down lactose, but it's not their favorite fuel.
 If glucose is around, they would much rather use that.
 Glucose requires fewer steps and less energy to break down than lactose.
 However, if lactose is the only sugar available, the E. coli will go right ahead and use it as an
energy source.
 To use lactose, the bacteria must express the lac operon genes, which encode key enzymes for
lactose uptake and metabolism.
 To be as efficient as possible, E. coli should express the lac operon only when two conditions are
met: a) When Lactose is available and b) Glucose is not available
 Changes in the lactose and glucose levels are detected and affects lac operon transcription

Structure of the lac operon

 The DNA of the lac operon contains (in order from left to right): CAP binding site, promoter,
operator, promoter and structural genes
 Promoter site (Plac) is found between the promoter and structural genes and is the binding site for
RNA polymerase, the enzyme that performs transcription
 Operator site (Olac) overlaps with promoter and is the negative regulatory site bound on by
the lac repressor protein; inducer binding site.
 When the lac repressor is bound, RNA polymerase cannot bind to the promoter or progress
downstream and start transcription.
 Catabolite Activator Protein (CAP) binding site is a positive regulatory site that is bound by
CAP.
 When CAP is bound to this site, it promotes transcription by helping RNA polymerase bind to
the promoter.
 Structural genes include lacZ gene, lacY gene, and lacA gene.
 The 3 genes codes for 3 key enzymes involved in lactose metabolism; Galactoside Permease or
lactose permease (Transports lactose into the cell across the cell membrane), Galactosidase
(Hydrolyzes lactose to glucose and galactose), and Thiogalactoside Transacetylase (initiate
metabolism), respectively.
 A lacI gene that codes for the lac repressor protein.
 The lacI gene has its own promoter (PlacI) that binds RNA polymerase and leads to transcription
of lac repressor mRNA and hence the production of lac repressor protein monomers.
  Four identical repressor monomers come together to form the active tetramer which can bind
tightly to the lac operator site, Olac to inhibit transcription of structural genes

Lac operon regulatory proteins

 The lac operon genes are expressed only when lactose metabolism is needed (when lactose is
present and glucose is absent).
 In addition to the three genes, the lac operon also contains a number of regulatory DNA
sequences.
 These are regions of DNA to which particular regulatory proteins can bind and regulate its
transcription
 Two regulators turn the operon "on" and "off" in response to lactose and glucose levels
 The two regulators involved are the lac repressor (acts as a lactose sensor) and catabolite
activator protein (CAP) (acts as a glucose sensor).
 These proteins bind to the DNA of the lac operon and regulate its transcription based on lactose
and glucose levels.
 The activator protein CAP, when bound to a molecule called cAMP, binds to the CAP binding
site and promotes RNA polymerase binding to the promoter, it promotes transcription.
 The lac repressor protein binds to the operator and blocks RNA polymerase from binding to
the promoter and transcribing the operon.

The role of lac repressor

 The lac repressor is a protein that represses (inhibits) transcription of the lac operon.
 It does this by binding to the operator, which partially overlaps with the promoter.
 When bound, the lac repressor gets in RNA polymerase's way and keeps it from transcribing the
operon.
 When lactose is not available (Glucose Present), the lac repressor binds tightly to the
operator, preventing transcription by RNA polymerase.
 When lactose is present (Glucose Absent), Allolactose binds to the lac repressor and makes it
lose ability to bind let’s go of the operator, clearing the way for RNA polymerase to transcribe
the operon.
 When lactose molecules are available, some will be converted to allolactose inside the cell.
 Allolactose binds to the lac repressor and makes it change shape so it can no longer bind DNA.
 Allolactose is an example of an inducer, a small molecule that triggers expression of a gene or
operon.
 The lac operon is considered an Inducible Operon because it is usually turned off (repressed) in
the presence of the lac repressor, but can be turned on in the presence of the inducer allolactose.
The role of catabolite activator protein (CAP)

 When lactose is present, the lac repressor loses its DNA-binding ability.
 This clears the way for RNA polymerase to bind to the promoter and transcribe
the lac operon.
 However, RNA polymerase alone does not bind very well to the lac operon promoter.
 It might make a few transcripts, but it won't do much more unless it gets extra help
from catabolite activator protein (CAP).
 CAP binds to a region of DNA just before the lac operon promoter and helps RNA polymerase
attach to the promoter, driving high levels of transcription.
 Cyclic adenosine monophosphate (cAMP) is a "hunger signal" made by E. coli when glucose
levels are low.
 When glucose levels are low, cAMP is produced.
 The cAMP attaches to CAP, forming CAP- cAMP complex
 cAMP binds to CAP, changing its shape and making it able to bind DNA and promote high
transcription
 The CAP in the CAP- cAMP complex helps RNA polymerase bind to the promoter, resulting in
high levels of transcription.
 Without cAMP, CAP cannot bind DNA and is inactive.
 When glucose levels are high, no cAMP is made. CAP cannot bind DNA without cAMP, so
transcription occurs only at a low level.
 Thus, the lac operon can only be transcribed at high levels when glucose is absent.
 This strategy ensures that bacteria only turn on the lac operon and start using lactose after they
have used up the entire preferred energy source (glucose).

IN CONCLUSION
 Glucose present, lactose absence: No transcription of the lac operon occurs. That's because
the lac repressor remains bound to the operator and prevents transcription by RNA
polymerase. Also, cAMP levels are low because glucose levels are high, so CAP is inactive
and cannot bind DNA.
Glucose present, lactose absence: No transcription of the lac operon occurs. That's because
the lac repressor remains bound to the operator and prevents transcription by RNA
polymerase. Also, cAMP levels are low because glucose levels are high, so CAP is inactive
and cannot bind DNA.
 Glucose present, lactose present: Low-level transcription of the lac operon occurs.
The lac repressor is released from the operator because the inducer (allolactose) is present.
cAMP levels, however, are low because glucose is present. Thus, CAP remains inactive and
cannot bind to DNA, so transcription only occurs at a low, leaky level.
 Glucose present, lactose present: Low-level transcription of the lac operon occurs.
The lac repressor is released from the operator because the inducer (allolactose) is present.
cAMP levels, however, are low because glucose is present. Thus, CAP remains inactive and
cannot bind to DNA, so transcription only occurs at a low, leaky level.
 Glucose absent, lactose absence: No transcription of the lac operon occurs. cAMP levels are
high because glucose levels are low, so CAP is active and will be bound to the DNA.
 However, the lac repressor will also be bound to the operator (due to the absence of
allolactose), acting as a roadblock to RNA polymerase and preventing transcription.
 Glucose absent, lactose present: Strong transcription of the lac operon occurs.
The lac repressor is released from the operator because the inducer (allolactose) is present.
cAMP levels are high because glucose is absent, so CAP is active and bound to the DNA.
CAP helps RNA polymerase bind to the promoter, permitting high levels of transcription.