QUESTIONS

1. Explain about central dogma of molecular biology. [7.5]

2. Differentiate between riboswitch and aptamers.[2.5]
3. Differentiate between chromosome, chromatin and chromatid.[2.5]
4. How RNA helps in gene regulation?[2.5]
5. Write about argonaute proteins.[2.5]
6. Write about positional effect for a gene.[2.5]
1. Explain about central dogma of molecular biology. [7.5]

The central dogma describes the flow of genetic information from DNA to RNA to protein. It deals with the
detailed residue-by-residue transfer of sequential information and states that such information cannot be
transferred back from protein to either protein or nucleic acid. The three major players in central dogma are
DNA and RNA, which are made up of nucleic acids and proteins which are made of amino acids. The
information starts at most basic level, stored as DNA.

Generally the genetic information flows in three pathways:

• DNA to DNA (replication)

• DNA to RNA (transcription)and
• RNA to proteins (translation)

I. DNA replication(DNA to DNA):

The information starts at most basic level, stored as DNA, which can be restored as DNA when DNA
copies itself in a process called replication. Replication is the basis for biological inheritance. The
enzyme DNA polymerase copies a single parental double-stranded DNA molecule into two daughter
double-stranded DNA molecules. During replication process; helicase enzyme unwinds the superhelix
as well as double strand of DNA to create a replication fork. Then DNA polymerase III reads the
template strand to create a daughter strand of DNA.

This process generally takes place during S phase of cell cycle.

II. Transcription (DNA to RNA):

The central dogma suggests that DNA contains the information needed to make all of our proteins, and
that RNA is a messenger that carries this information to the ribosome. Transcription is the process of
creating a complementary RNA copy of a sequence of DNA. During transcription, a DNA sequence is
read by RNA polymerase, which produces a complementary, antiparallel RNA strand. Unlike DNA
replication, transcription results in an RNA complement that substitutes the RNA uracil (U) in all
instances where the DNA thymine (T) would have occurred. Transcription is the first step in gene
expression. The stretch of DNA transcribed into an RNA molecule is called a transcript. Some
transcripts are used as structural or regulatory RNAs, and others encode one or more proteins. If the
transcribed gene encodes a protein, the result of transcription is messenger RNA (mRNA), which will
then be used to create that protein in the process of translation.

III. Translation(RNA to Protein):

Translation is the process by which mRNA is decoded and translated to produce a polypeptide
sequence, otherwise known as a protein. This method of synthesizing proteins is directed by the mRNA
and accomplished with the help of a ribosome, a large complex of ribosomal RNAs (rRNAs) and
proteins. In translation, a cell decodes the mRNA’s genetic message and assembles the brand-new
polypeptide chain. Transfer RNA, or tRNA, translates the sequence of codons on the mRNA strand.
The main function of tRNA is to transfer a free amino acid from the cytoplasm to a ribosome, where it
is attached to the growing polypeptide chain. tRNAs continue to add amino acids to the growing end of
the polypeptide chain until they reach a stop codon on the mRNA. The ribosome then releases the
completed protein into the cell.
In some exceptional cases the flow of genetic information occurs in different order.

Special transfers of biological sequential information:

➢ Reverse transcription:
Reverse transcription is the transfer of information from RNA to DNA (the reverse of normal
transcription). This is known to occur in the case of retroviruses, such as HIV, as well as
in eukaryotes, in the case of retrotransposons and telomere synthesis. It is the process by
which genetic information from RNA gets transcribed into new DNA.

➢ RNA replication:
RNA replication is the copying of one RNA to another. Many viruses replicate this way. The
enzymes that copy RNA to new RNA, called RNA-dependent RNA polymerases, are also
found in many eukaryotes where they are involved in RNA silencing

➢ Direct translation from DNA to protein:

Direct translation from DNA to protein has been demonstrated in a cell-free system (i.e. in a
test tube), using extracts from E. coli that contained ribosomes, but not intact cells. These cell
fragments could synthesize proteins from single-stranded DNA templates isolated from other
organisms (e,g., mouse or toad), and neomycin was found to enhance this effect. However, it
was unclear whether this mechanism of translation corresponded specifically to the genetic
code.
2. Differentiate between riboswitch and aptamers.[2.5]
Riboswitches are elements commonly found in the 5′-untranslated region (UTR) of mRNAs that exert
their regulatory control over the transcript in a cis-fashion by directly binding a small molecule ligand.
The typical riboswitch contains two distinct functional domains. The effector molecule is recognized by
an aptamer domain, which adopts a compact three-dimensional fold to scaffold the ligand binding
pocket.

Aptamers are small single-stranded nucleic acids that fold into a well-defined three-dimensional
structure. They show a high affinity and specificity for their target molecules and inhibit their biological
functions. Aptamers assume a variety of shapes due to their tendency to form helices and single-stranded
loops.
The aptamer directly binds the small molecule, and the expression platform undergoes structural changes
in response to the changes in the aptamer. The expression platform is what regulates gene expression.
Aptamer domain along with expression platform makes up the riboswitch.
 3. Differentiate between chromosome, chromatin and chromatid.[2.5]

Chromatin is a mass of genetic material composed of DNA and proteins that condense to form chromosomes
during eukaryotic cell division. In the nucleus, chromatin exists as euchromatin or heterochromatin.
Euchromatin is in less compact form exposing more DNA and allows transcription and replication process to
take place. While, heterochromatin is tightly packed, not allowing transcription to occur.

Chromosomes are single-stranded groupings of condensed chromatin. During the cell division processes of
mitosis and meiosis, chromosomes replicate to ensure that each new daughter cell receives the correct number
of chromosomes. A duplicated chromosome is double-stranded and has the familiar X shape. The two strands
are identical and connected in a central region called the centromere.

A chromatid is either of the two strands of a replicated chromosome. Chromatids connected by a centromere are
called sister chromatids. At the end of cell division, sister chromatids separate becoming daughter chromosomes
in the newly formed daughter cells.
4. How RNA helps in gene regulation?[2.5]

RNA interference (RNAi) is a biological process in which RNA molecules inhibit gene expression or
translation, by neutralizing targeted mRNA molecules. Double-stranded RNA-mediated interference (RNAi)
is a simple and rapid method of silencing gene expression in a range of organisms. The silencing of a gene is
a consequence of degradation of RNA into short RNAs that activate ribonucleases to target homologous
mRNA.

There are several types of small regulatory RNAs:

Small double stranded RNA(siRNAs) are derived from longer double stranded RNAs that are either
produced in cell or introduced experimentally.

MicroRNAs (miRNAs) are another type of small RNA. Most miRNAs come from RNAs that are
transcribed in nucleus which then folds and are processed before transporting to cytoplasm as double
stranded precursor miRNAs.

The double stranded precursors of miRNAs and siRNAs binds to dicer which is an endonuclease protein
which cuts RNA in short segments. Most siRNAs and miRNAs are approximately 21 nucleotides long. The
short double stranded RNAs now bind argonaute protein. One strand of the RNA remains bound to the
protein, known as guide strand. The combination of argonaute with RNA and other proteins is called RISC
(RNA-induced silencing complex). The siRNAs direct RISC to bind to specific RNAs. The targeting is
precise because it is determined by basepairing between siRNA and target messenger RNA. Once bound,
argonaute catalyses the cleavage of messenger RNA which will then be degraded.

miRNAs also guide RISC to mRNAs. Only a part of miRNAs, known as seed, bind to the mRNA. This
imprecise pairing allows miRNA to target hundreds of endogenous mRNAs. The binding of miRNAs can
lead to degradation of mRNA or it being silenced.
 5. Write about argonaute proteins.[2.5]

Argonaute proteins are highly specialized small-RNA-binding modules and are considered to be the key
components of RNA-silencing pathways. Argonaute family proteins can be divided into AGO and PIWI
proteins. Both types of Argonaute proteins bind 21–35 nucleotide long small RNA guides whose sequence
identifies the genes to be silenced. Argonaute–small-RNA complexes can repress the transcription of genes,
target mRNAs for site-specific cleavage or general degradation, or block mRNA translation into protein. AGO
proteins bind ~21 nucleotide small interfering RNAs (siRNAs) and 21–23 nucleotide microRNAs (miRNAs).
Both siRNAs and miRNAs are cut from double-stranded RNA precursors by RNase III enzymes such as Dicer.
AGO proteins are essential for development and differentiation, and in most plants and animals, defend cells
against viral infection. In contrast, PIWI proteins bind 23–30 nucleotide PIWI-interacting RNAs (piRNAs),
whose production does not appear to involve double-stranded RNA or Dicer. piRNAs are unique to animals,
where they repress transposon expression and ensure the successful production of sperm and eggs

An Argonaute protein plus its small RNA guide compose the RNA-induced silencing complex (RISC). RISC
complexes can also contain additional proteins thought to extend the functions of Argonautes or to direct RISC
to specific sub-cellular locations. The simplest, and likely ancestral, Argonaute function is endonucleolytic
cleavage of its RNA target at a single phosphodiester bond. The structure of Argonaute ensures that the bond
cleaved always lies between the target nucleotides paired to the tenth and eleventh nucleotides of the guide
RNA.
 6. Write about positional effect for a gene.[2.5]

A positional effect is defined as a deleterious change in the level of gene expression brought about by a change
in the position of the gene relative to its normal chromosomal environment, but not associated with an
intragenic mutation or deletion. Thus, the transcription unit and minimal promoter of the rearranged gene are
expected to remain intact.

There are broadly three categories of factors determining correct gene expression:

(i) The promoter region:

The site where the basal transcription machinery loads onto the DNA and initiation of transcription
occurs;
(ii) Enhancer/silencer elements:
Short DNA regions containing binding sites for transcription factors which are often tissue-specific and
aid in assembly of the transcription machinery on the promoter, independently of orientation and
position with respect to the promoter

(iii) The local chromatin environment of the gene locus:

Enhancers and promoters can only function correctly in a permissive chromatin environment, which
makes them accessible for protein interaction

Chromosomal rearrangements frequently lead to alteration of the gene's environment and this may be reflected
in a change of expression.
• The chromosomal rearrangement could separate the promoter/transcription unit from an essential
distant regulatory element, thus removing the effect of this regulator on the gene. The absence of the
enhancer element leads to reduction or absence of transcription from the gene in the appropriate tissue.
Alternatively, if the element is involved in silencing the gene in a particular tissue, then its removal
through rearrangement could lead to inappropriate activation of the gene.
• A rearrangement may also juxtapose the gene with an enhancer element from another gene, again
leading to inappropriate gene expression.
• The translocation could place the gene and its regulatory elements next to a second gene present at the
site of translocation. Competition for the regulatory element between the disease gene and second gene
may result in a reduction in expression levels
• Positional Effect Variegation (PEV) can occur when a chromosomal rearrangement causes the
juxtaposition of a euchromatic gene with a region of heterochromatin. The heterochromatin DNA
organization is thought to spread into the juxtaposed euchromatic region, thereby silencing the nearby
gene in a stochastic manner.
REFERENCE
1

➢ https://www.yourgenome.org/facts/what-is-the-central-dogma
➢ https://www.khanacademy.org/test-prep/mcat/biomolecules/amino-acids-and-
proteins1/v/central-dogma-of-molecular-biology-2

➢ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3098680/
➢ https://www.basepairbio.com/what-is-an-aptamer/
➢ https://www.ncbi.nlm.nih.gov/pubmed/19319884
➢ https://www.youtube.com/watch?v=LNV1ipK6Cz0

➢ https://www.thoughtco.com/chromatin-373461
➢ https://www.khanacademy.org/science/high-school-biology/hs-reproduction-and-cell-
division/hs-chromosome-structure-and-numbers/v/chromosomes-chromatids-chromatin-etc
➢ https://www.difference.wiki/chromatin-vs-chromatid/

➢ https://www.youtube.com/watch?v=cK-OGB1_ELE
➢ https://mmbr.asm.org/content/67/4/657
➢ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2727154/
➢ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5085744/

➢ https://www.cell.com/current-biology/pdf/S0960-9822(11)00547-1.pdf
➢ http://jcs.biologists.org/content/123/11/1819

➢ https://academic.oup.com/hmg/article/7/10/1611/635945
➢ https://pdfs.semanticscholar.org/8ed3/c9a49ed0cae340640a62e7a3957fd08fc7c8.pdf