Name : Ndumiso

Surname : Ndawonde

Student number : 60516364

Examination period : May /June 2020

ID number : 9704175437084

Module : Molecular Genetics, BCH3703

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Question 1

1.1

PDB code for Names of the Secondary Tertiary or Sections in the

the molecule molecules in structural quaternary study material
the structure elements structure of the relating to this
present molecule molecule
1KX3 Nucleosome Helices and Tertiary Section 1.2,
core particle strands folded elements nucleosome.
into domains, arranged in Has DNA called
forming histones, which
quaternary
functional allows for
structure. It is
subunits. production of
hereto 8-mer,
compact storage
therefore a
of genetic
quaternary. material in
eukaryotes.
5IYC Human core- Helices and beta Tertiary Section 2.4,
PIC. strands folded structure. transcription
into domains. factors binding
to DNA and
regulating
transcription
initiation.
Transcription
factors plays a
huge role in the
activation of
domains that
allows them for
interaction with
proteins.

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Question 2

DNA melting is dissociation of the double stranded DNA helix into single coils, accomplished by
heating double stranded DNA. This depends on the number of hydrogen bonds holding
complementary strands. There are two hydrogen bonds between adenine and thymine and
three bonds between guanine and cytosine. The principle behind this is that the more G –C in
the sequence the higher the temperature needed to melt a DNA fragment.

The method used to differentiate DNA strands using DNA melting experiments is to subject the
product to a temperature gradient into the presence of intercalating dye, which are chemicals
that emits light when bound to double strand DNA. In a melting experiment DNA is mixed with
an intercalating dye and fluorescence emitted by this mix is monitored as the sample is slowly
heated. The outcome of the experiment is a curve displaying fluorescence changes emitted by
the sample over a range of temperature that the DNA was subjected to, commonly known a
melting profile. At the beginning of the melting experiment the temperature is low and all
DNA strands in the sample are double stranded. Thus, we observe high levels of
florescence from the sample (DNA strand 1. We continue to observe high levels of
fluorescence, as the temperature increases up to the point, where all hydrogen bonds
within the DNA strand are broken and the amount of double stranded DNA strands
decreases, observing a sharp decrease in the detected fluorescence level (DNA strand
2). At a high temperature there is no double stranded DNA strand in the sample and the
fluorescence levels are close to 0 (DNA strand 3). The temperature at which we
observe the sharp drop in the fluorescence depends on the number of hydrogen bands
in the analyzed DNA strands.

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Question 3

All the information necessary for the correct folding of a protein is present in its amino
acid sequence. The order and type of amino acid specify certain structural elements.
There are key amino acids that are found to form alpha helices and beta strands.
Chaperones are the name given to proteins that block undesirable interactions in a
polypeptide during folding. There are many different types of chaperones. Some are
small proteins that bind hydrophobic patches on the newly synthesised polypeptide until
the rest of the polypeptide is synthesised and the correct regions can unite to form the
fully functional protein. Without these chaperones, proteins can misfold, producing
ordered aggregates of protein. These aggregates accumulate in cells and can cause
diseases such as Alzheimer’s disease. The correct formation of disulphide bonds is
facilitated in eukaryotes through proteins called protein disulphide isomerases (PDIs).
These proteins catalyse the correct disulphide formation and the rearrangement of
incorrect disulphide bonds that might have formed during folding.

Besides PDIs, there are other groups of proteins that assist in folding. These are
peptidyl prolyl cis-trans isomerases and molecular chaperones. Peptidyl prolyl cis-trans
isomerases catalyse the switching of X-proline peptide bonds between cis and trans
conformations. Molecular chaperones, often known as heat shock proteins, function to
bind and shield hydrophobic regions on a polypeptide until it can interact with the
correct regions of the polypeptide for folding to the functional native state. There are
many different types of molecular chaperones, such as trigger factors and
nucleoplasmins, which help with the proper assembly of nucleosomes. Many proteins
are covalently modified through the attachment of chemical groups. These modifications
are called posttranslational modifications and can involve the attachment of acetyl,
hydroxyl, phophoryl and methyl groups.

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Question 4

Chromatin structure plays a major role in controlling eukaryotic gene expression. DNA
that is densely packaged into chromatin is less susceptible to cleavage by the
nonspecific DNA-cleaving enzyme DNase I. Regions adjacent to genes that are being
transcribed are more sensitive to cleavage by DNase I than are other sites in the
genome, suggesting that the DNA in these regions is less compacted than it is
elsewhere and more accessible to proteins. Some sites, usually within 1 kb of the start
site of an active gene, are exquisitely sensitive to DNase I and other nucleases. Genes
required for galactose utilization in yeast are usually activated by a transcription factor
called GAL4, which recognizes DNA-binding sites with two 5’- CGG-3’ sequences on
complementary strands separated by 11 base pairs. Approximately 4000 potential
GAL4 binding sites of the form 5’- CGG (N)11CCG-3’ are present in the yeast genome,
but only 10 of them regulate genes necessary for galactose metabolism. This is
addressed through the use of a technique called chromatin immunoprecipitation. GAL4
is first cross-linked to its DNA-binding sites in chromatin. The DNA is then fragmented
into small pieces, and antibodies to GAL4 are used to isolate the chromatin fragments
containing GAL4. The cross-linking is reversed, and the DNA is isolated and
characterized, resulting in only approximately 10 of the 4000 potential GAL4 sites which
are occupied by GAL4 when the cells are growing on galactose, presumably by the
local chromatin structure. GAL4 is thereby prevented from binding to sites that are
unimportant in galactose metabolism. These lines of evidence and others reveal that
chromatin structure is altered in active genes compared with inactive ones.

The degree of methylation of DNA provides another mechanism, in addition to

packaging with histones, for inhibiting gene expression inappropriate to a specific cell
type. Carbon 5 of cytosine can be methylated by specific methyltransferases. About
70% of the 5’- CpG-3’ sequences (“p” for phosphate residue in the DNA backbone) in
mammalian genomes are methylated. However, the distribution of these methylated
cytosine varies, depending on the cell type. . The relative absence of 5-methylcytosines
near the start site is referred to as hypomethylation. The methyl group of 5-m ethyl
cytosine protrudes into the major groove where it could easily interfere with the binding

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of proteins that stimulate transcription. Many CpG sequences have been converted into
TpG through mutation by the deamination of 5-methylcytosine to thymine. However,
sites near the 5’ ends of genes have been maintained because of their role in gene
expression.

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Question 5

Transcription factors are the proteins that are key to gene regulation in both prokaryotes
and eukaryotes. In eukaryotes, the expression of each gene can be controlled by
several transcription factors and the expression of genes can be co-ordinated by each
gene having similar transcription factor binding sites. Transcription factors in eukaryotes
can act in the following three ways that is, by interacting directly with RNA polymerase,
by interacting with proteins associated with RNA polymerase, by modifying the
chromatin structure. Eukaryotic transcription factors usually consist of several domains,
including at least a DNA-binding domain and at least one domain that helps in the
activation of transcription. The most important aspects of transcription factors are the
DNA motifs they commonly bind to, transcription factor activation domains that allow
them to interact with proteins, the ability of transcription factors to form complexes
containing multiple transcription factors, and stretches of DNA sequence that act as
enhancers of gene expression when transcription factors are bound to them.

Three common motifs found in DNA-binding proteins are homeodomain, basic-leucine

zipper protein and Cys2His2 zinc-finger domains. Homoedomain are very similar to
prokaryotic helix-turn-helix proteins. They form heterodimeric structures, which are able
to recognize asymmetric DNA sequences. Basic-leucine zipper (bZip) proteins consist
of a pair of long alpha helices. The initial part of the alpha helix lies in the major groove
of the DNA, and the second part forms a coil with its partner. These structures are
stabilized by precisely spaced leucine residues. Cys2His2 zinc-finger domains comprise
random sets of small domains which each bind a zinc ion owing to conserved cysteine
and histidine residues. These domains form a long string that follows the major groove
of DNA. Zinc fingers allow transcription factors to be in contact with long stretches of
DNA at one time.

An important target of activators is a complex known as the mediator, which contains 25

to 30 subunits and has been conserved in all eukaryotes, from yeast to human beings.
The mediator acts as a bridge between transcription factors and promoter-bound RNA
polymerase II. Activation domains may be acidic, hydrophobic, glutamine rich or proline
rich. Despite a lack of sequence similarity, common features among activation sites

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include being redundant, meaning parts of the domain can be deleted without loss of
function. They are also modular and can bind to a variety of DNA-binding domains while
still being able to activate transcription. They also act synergistically.

Several transcription factors join with RNA polymerase II to form the basal transcription
complex. The basal transcription complex initiates transcription at a low frequency. Few
eukaryotic transcription factors act on their own, but instead recruit other proteins to
form complexes that interact with transcriptional machinery. This leads to combinational
control, where a single factor can have multiple different effects depending on the
different proteins with which it interacts.

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Question 6

6.1. Riboswitches are special secondary structures of the nRNA transcript that
regulated trp operon. They are called cis-acting elements because they act on the same
molecule. An example of riboswitches is the synthesis of riboflavin in bacillus subtilis.
When there are high concentrations of Flavin Mono Nucleotide, a key intermediate in
riboflavin synthesis, FMN binds a conformation of the RNA transcripts, resulting in the
stabilization of a hairpin structure that causes premature termination of transcription. If
FMN levels are low, then it cannot bind the RNA transcript and an alternative
conformation of the transcript forms, allowing complete transcription of the transcript.

6.2. Yes I think the RNA sequence could be part of a riboswitch mechanism because it
does satisfy the RNA-RNA interaction that is at the translational level, effector binding to
the aptamer domain occludes the Shine-Dalgarno sequence that recruits the
30S subunit by pairing with a site near the 3′-end of the 16S ribosomal RNA. Thus,
riboswitch contain the essential features to efficiently function in the hypothesized RNA
world.

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Question 7

Location of Modification Modification Modification

modification on description of description for description for
RNA molecule rRNA tRNA mRNA
5’ end 5’splice site. 5’ leader cleavage by 5’ cap & S-
RNase P. adenosylmethionine
3’ end 3’ splice site. 3’ trailer removed and Poly(A) polymerase
CCA added by ads about 250
nucleotidyltransferase adenylate residues
5enzyme to the 3’ end,
producing a poly(A)
tail
Changes to the Cleavage to Splicing in Transesterification
internal produce 18S, 28S eukaryotes. The reactions that take
sequences of and 5.8S rRNA enzymes involved in place to produce
the RNA molecules; this splicing are the final mature
molecule modifications made endonuclease and a mRNA. Also
to the transcript by ligase. interacts with small
small nuclear nuclear RNAs and
ribonucleoproteins protein complexes
(SnoRNPs), which called snRNPs
consist of one (small nuclear
snoRNA and ribonucleoprotein
several proteins. particles) in a
Methylation of the complex called the
sugar backbone spliceosome, which
catalyses the
splicing reaction.

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Question 8

prokaryotic eukaryotic
No cap on mRNA 5’ end of mRNA recognized by cap
Start codon is next AUG after ribosome No ribosome-binding site so first AUG in
binding site mRNA is used
First amino acid is formyl-Met First Met is unmodified
70S ribosomes made of: 30S and 50S 80S ribosomes made of: 40S and 60S
subunits subunits
Small 30S subunit: 16S rRNA and 21 Small 40S subunit: 18S rRNA and 33
proteins proteins
Large 50S subunit: 23S and 5S rRNA Large 60S subunit: 28S, 5.8S and 5S
plus 31 proteins rRNA plus 49 proteins
Elongation factors: EF-T (2 subunits) and Elongation factors: eEF1 (3 subunits) and
EF-G eEF2
Three initiation factors: IF1, IF2 and IF3 Multiple initiation factors: eIF2 (3
subunits), eIF3, eIF4 (4 subunits), eIF5
polycistronic mRNA Monocistronic mRNA
Coupled transcription and translation No coupled transcription and translation
for nuclear genes

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Question 9

1. DNA topoisomerases = helps relieve the stress on DNA when unwinding by

causing breaks and then resealing the DNA.
2. polymerase I = exonuclease activity removes RNA primer and replaces with
newly synthesized DNA
3. DNA polymerase II = responsible for DNA repair
4. DNA polymerase III = primary enzyme of DNA synthesis, catalyses elongation of
both leading and lagging strands.
5. DNA ligase = seals the gaps between okazaki fragments to create one
continuous DNA strand

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Question 10

10.1. The aim of the overall research was to purify the DNA dependent enzyme, RNA
polymerase from clostridium acetobutylicum to homogeneity and characterized.

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Question 11

Purification should yield a sample containing only one type of molecule, the protein in which the
researcher is interested in. A protein can be purified by subjecting the impure mixture of the
starting material to a series of separations based on physical properties such as size and
charge. To monitor the success of this purification, the researcher needs a test, called an assay,
for some unique identifying property of the protein. A positive result on the assay indicates that
the protein is present. For enzymes, which are protein catalysts, the assay usually measures
enzyme activity that is the ability of the enzyme to promote a particular chemical reaction.
Several thousand proteins have been purified in active form on the basis of such characteristics
as solubility, size, charge, and specific binding affinity. At each step in the purification, the
preparation is assayed and its specific activity is determined.

Salting out is where most proteins are less soluble at high salt concentrations. The salt
concentration at which a protein precipitates differs from one protein to another. Hence, salting
out can be used to fractionate proteins. Salting out is also useful for concentrating dilute
solutions of proteins, including active fractions obtained from other purification steps. Dialysis, is
where proteins can be separated from small molecules such as salt by dialysis through a
semipermeable membrane, such as a cellulose membrane with pores. The protein mixture is
placed inside the dialysis bag, which is then submerged in a buffer solution that is devoid of the
small molecules to be separated away. Molecules having dimensions significantly greater than
the pore diameter are retained inside the dialysis bag. Smaller molecules and ions capable of
passing through the pores of the membrane diffuse down their concentration gradients and
emerge in the solution outside the bag. This technique is useful for removing a salt or other
small molecule from a cell fractionate, but it will not distinguish between proteins effectively

Gel-filtration chromatography is where more discriminating separations on the basis of size can
be achieved by this technique, also known as molecular exclusion chromatography. The sample
is applied to the top of a column consisting of porous beads made of an insoluble but highly
hydrated polymer such as dextran or agarose or polyacrylamide. Sephadex, Sepharose, and
Biogel are commonly used commercial preparations of these beads, which are typically 100 mm
in diameter. Small molecules can enter these beads, but large ones cannot. The result is that
small molecules are distributed in the aqueous solution both inside the beads and between
them, whereas large molecules are located only in the solution between the beads. Affinity
chromatography. Affinity chromatography is another powerful means of purifying proteins that

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is highly selective for the protein of interest. This technique takes advantage of the high affinity
of many proteins for specific chemical groups

Ion-exchange chromatography. To obtain a protein of high purity, one chromatography step is

usually not sufficient, because other proteins in the crude mixture will likely co-elute with the
desired material. Additional purity can be achieved by performing sequential separations that
are based on distinct molecular properties. This procedure is also referred to as cation
exchange to indicate that positively charged groups will bind to the anionic beads. Positively
charged proteins (cationic proteins) can be separated by chromatography on negatively charged
carboxymethylcellulose columns. Conversely, negatively charged proteins can be separated by
anion exchange on positively charged diethylaminoethylcellulose (DEAE-cellulose) columns.
affinity chromatography can be effectively used to isolate a protein that recognizes group X by
covalently attaching X or a derivative of it to a column; adding a mixture of proteins to this
column, which is then washed with buffer to remove unbound proteins; and eluting the desired
protein by adding a high concentration of a soluble form of X or altering the conditions to
decrease binding affinity.

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Question 12

Two techniques used are gel electrophoresis and Mass spectrometry. Gel electrophoresis, the
ability of molecules with a net charge to migrate within an electric field is termed
electrophoresis. Nucleic acids and proteins have an intrinsic charge, which depends on the
buffer conditions in which they are placed, and they migrate in solution if an electric field is
applied to the solution. Electrophoresis is not carried out in solution, but rather through a porous
gel or even solid support media like paper. The support medium is inert and serves as a sieve, it
aids in separating the molecules. The intrinsic shape of molecules also has an impact on their
separation. Polyacrylamide is predominantly used for the separation of proteins, but agarose
can also be used. Agarose is often used in the separation of DNA, but polyacrylamide can be
used for short DNA or RNA molecules. The choice of support media depends on the size of the
molecules which need to be separated. Proteins have a net charge that depends on the buffer
conditions in which they are placed. If they are placed in buffer with an equivalent pH to their
isoelectric point, they have a net zero charge. Above and below their isoeletric point they are
positively or negatively charged respectively. During isoelectric focusing, this property is
exploited. Proteins are first separated in a support medium according to their charge. The
proteins migrate to a region of pH that corresponds to their isoeletric point value and then they
are separated on the basis of size, using SDS-PAGE (sodium dodecyl sulphate polyacrylamide
gel electrophoresis). In SDS-PAGE the protein interacts with SDS, which is a strong denaturant.
It removes the intrinsic shape and charge of the protein, coating the now linear protein in a net
negative charge. This means that all proteins migrate strictly owing to their molecular weight.
Small proteins move rapidly through the gel, while larger proteins move slowly through the gel.
Once the proteins have been separated, the proteins are stained so that they are visible in the
gel.

Mass spectrometry allows researchers to identify proteins without the use of antibodies.
Molecules are first converted to the gaseous state. Different types of ion sources are used to do
this – matrix-assisted laser desorption/ionization (MALDI) and electrospray ionisation (ESI) are
the two main methods used. In the MALDI method, a laser excites and vaporises the molecules.
With ESI a solution of the molecules is passed through a charged nozzle and the charged
droplets then emerge into a chamber under low pressure, which results in evaporation. Once
ionised, the detector has to measure the mass of the molecules. This is done with the
instrument measuring the mass-to-charge ratio of the different molecules. Different types of
detectors or mass analysers exist, but the time-of-flight (TOF) is the simplest. TOF detectors

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measure the time it takes the charged molecule to move through a charged chamber. Molecules
of similar charge but different masses move at different rates. Mass spectrometry can be used
to determine the primary structure or sequence of a protein. This will confirm that a purified
protein is the one of interest and that no mutations have occurred.

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References

https://jb.asm.org/content/173/6/2120

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC207749/

https://www.rcsb.org/structure/1KX3

https://www.rcsb.org/structure/1A0I#entry-history

https://www.wwpdb.org/

Berg, JM, Tymoczko, JL, Gatto Jr, GJ & Stryer, L. 2015. Biochemistry. 8th edition. New York:
WH Freeman/Macmillan.

BCH3703 study guide, UNISA, 2020.

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