RNA
RNA or Ribonucleic Acid is a crucial molecule that translates the genetic code from
DNA into proteins, driving cellular activities. Unlike DNA's double helix, RNA is normally
single stranded, containing a sugar called ribose and the bases, adenine, guanine,
cytosine & uracil instead of thymine. RNA serves as a messenger, carrying information
from DNA for protein synthesis & plays various roles in gene expression and regulation
Primary function of RNA is to convert the genetic instructions found in DNA into the
proteins that drive cellular processes. RNA is a polymer of ribonucleotides held together
by phosphodiester bridges.
History
Nucleic acids now discovered in nucleus of eukaryotic cells. It was later discovered that
prokaryotic cells which do not have a nucleus but contain Nucleic Acid. It does not
follow Chargaff's rule. It is mainly cytoplasmic but also present in nucleus.
The base content varies from 100-5000. The size is variable.
Single Stranded Helix – Its nature allows it to fold into complex shapes, enabling it to
participate in a wide range of cellular functions, from catalysis to regulation.
Nucleotides – consist of a sugar molecule (ribose), a phosphate group & one of 4
nitrogenous bases (A, G, C, U).
Ribose – Sugar in RNA differs from deoxy-ribose in DNA having an additional hydroxyl (-
OH) group. This small change makes RNA more reactive and less stable than DNA.
Phosphate – Connects the ribose sugars, the phosphate group forms the backbone of
the RNA strand, similar to DNA imparts a -ve charge to the molecule.
Nitrogenous base – Include Adenine, Guanine, Cytosine & Uracil. Uracil replaces
Thymine (T) found in DNA and pairs with Adenine during RNA synthesis and function.
Hydrogen Bonds – form between bases of the same molecule, folding it into complex
structures necessary for its various roles.
Major & Minor grooves – RNA structure also includes grooves where proteins and other
molecules can bind, influencing its function & regulation.
Susceptibility to alkali hydrolysis – Alkali can hydrolyse RNA to 2’-3’ cyclic diesters. This
is possible due to the presence of a hydroxyl group at 2’ position. DNA cannot be
subjected to alkali hydrolysis due to lack of this group. Orcinol color reaction – RNA can
be histologically identified by orcinol colour reaction due to presence of ribose.
Types of RNA
In all prokaryotic & eukaryotic organisms, three main classes of RNA molecules exist –
(i) Messenger (mRNA) – 5-10% (Cellular composition)
(ii) Transfer (tRNA) – 10-20%
(iii) Ribosomal (rRNA) – 50-80%.
Other – Small nuclear RNA (snRNA)
Micro RNA (miRNA)
Small interfering RNA (siRNA)
Heterogeneous nuclear RNA (hnRNA).
Messenger RNA (mRNA)
The mRNA is synthesized in the nucleus (in eukaryotes) as heterogeneous nuclear RNA
(hnRNA), on processing liberates the functional mRNA which enters the cytoplasm to
participate in protein synthesis. mRNA has high molecular weight and has short half-life
& show long mobile themes in a cell before broken down.
In general mRNA of eukaryotes is more stable with longer half life, compared to
prokaryotic mRNA.
Function –
➔ Carries the instructions from DNA to build proteins. The longer an mRNA lasts
(longer half-life), the more protein the cell can make from that single set of
instructions
➔ cell needs to respond quickly to changing conditions. A short half life allows the
cell to rapidly turn off protein production when that protein is no longer needed,
by simply degrading the existing instructions.
➔ Carrier of genetic code – mRNA acts as an intermediary, carrying a copy of
genetic instructions from DNA to the ribosomes
➔ Template for protein synthesis -It provides the blueprint for building proteins.
Ribosomes read the sequence of codons on the mRNA to assemble the correct
order of amino acids, forming a polypeptide chain.
➔ Link between nucleus and cytoplasm – It transfers information out of the
nucleus, where the genetic information is housed, to the ribosome in the
cytoplasm where proteins are made. This is crucial because DNA is too large to
leave the nucleus.
➔ Basis for therapeutics & vaccines. Modified mRNA sequence can be used to
treat diseases or create vaccines, as seen in some covid-19 vaccines, by
instructing the body to produce specific proteins.
Structure –
5’ cap
A mature mRNA is. Capped with a 5’-cap before the 5’ untranslated region. The 7-
methylguanine triphosphate associates with the 5’ end via 5’-5’ phosphate linkage. It
performs protection towards degeneration. The 5’ end of mature mRNA against
degeneration prevent hydrolysis of mRNA by 5’-exonuclease.
It involves in the recognition of mRNA for protein synthesis by binding to ribosome.
Poly (A) tail
The 3’ terminal end of mRNA contains a polymer of adenylate residues (20-250
nucleotides) known as poly (A) tail after the 3’ untranslated region. It provide stability to
mRNA thereby prevent it from the attack of 3’-exonuclease.
It helps in transportation of mRNA remotely within the nucleus into the cytoplasm.
Transfer RNA (tRNA) -
• tRNA (transfer RNA): Soluble RNA Molecule,(soluble in sol of 1m conc of
back) smaller than mRNA. Contains 71-80 nucleotides with a Molecular
weight of about 25KD. There are at 20 species of tRNAs Corresponding to
20 aa present in protein structure. It acts as the intermediary element
playing a significant role in the loading & transferring aa to the site of
protein synthesis i.e. ribosome. The tRNA first decodes the information or
nucleotide sequences carried by mRNA.
• The mRNA carries a code / nucleotide sequences of DNA and a tRNA is the
key to that code Carrying anticodone that can decode the genetic codons
of mRNA. A complementary occurs between triplet codons of mRNA with
anticodon of tRNA.
• tRNA is single stranded having 5'-3' end.
• tRNA is a highly folded structure due to which the nucleotide come closer
to each other & appears to be like double - stranded, etc.
• formation of stem-loop structure occurs in the tRNA due to abnormal base
pairs and no hydrogen bonding, in case of regular base pair & no H-bond
between them.
Structure
There are 3 structural configurations of tRNA.
Primary – tRNA possesses a linear structure consisting of 60-90 nucleotides. The
primary structure forms post-transcriptionally. The charged tRNA assembles the
amino acid in correct order where the carboxyl group attaches above the 3’-end. On
its 3’-end CCA group is present.
Secondary – The “Clover leaf model” is the most popular model to explain the 2°
structure of tRNA. Robert Holley gave it in 1968. According to clover leaf model, four
arms exist in the structure of tRNA & sometimes one additional arm can also be
present.
Acceptor DHU, Anticodon, T psi C arms,.
Acceptor's arm" - The 𝑎𝑎 attachment site. Consists of 7 paired nucleotides & 4 unpaired
bases. The acceptor 3' arm is a double helical stem having both 3' end & 5' end. The 3' end
consists of a CCA sequence or terminals ACC-sequence. The carbonyl group of
𝑎𝑎attaches with 3'-OH end. At the 5' end, guanine is present (Guan), Therefore it purely
refers to the attachment site for the activated 𝑎𝑎
DHU arm - Stands for Di-hydroxy uridine. Consists of 15-18 total bases, form which 7-9 are
modified bases & 4 unusual bases. It comprises of a 4 bp long stem & a loop with 9 unusual
bases including dihydroxy uridine. DHU arm is known as "Aminoacyl Synthetase binding
site" as it activates the aa synthesis by Synthesizing an enzymes. (aminoacyl tRNA
synthetase) - This enzyme promotes the specific and robust binding of aa to the tRNA with
the help of an ATP molecules. DHU plays a very crucial role in the stabilization of the tRNA
tertiary structure. There are two variable region present on both the sides of guanine
residue 𝛼 & 𝛽 variable areas.
Anticodon arm - It is the codon-recognition site. Consists of a stem which is 5 bp long & a
loop consists of 7 unpaired bases. In between the seven unpaired bases of anticodon loop;
there are three anticodons. The anticodons recognize the codon of mRNA and
complementarily binds to it. The anticodon in the loop decides the type of aa will attach to
the 3’ end of t-RNA. Therefore, the anticodon arm is an essential part of tRNA that
recognizes & reads the info carried by mRNA.
T psi C arm – Stands for Thymine, pseudouracil, cytosine. Consists of a stem 5bp long & a
loop with 4 unpaired bases. Due to presence of T psi C sequences in 5’ -3’ directions it is
named as T psi C arm. It also possesses or “Ribosomal recognition site” that plays a vital
role in binding tRNA with the ribosome.
Variable arm – additional arm present b/w the anticodon & T psi C arms – It is shortest
arm & its presence differs among species to species. The length of variable arm recognize
the enzyme translated for tRNA. It helps in stabilization of the t-RNA based on the presence
of a variable arm. 2 types: Type – I tRNA lacks the variable arm 3-5 bp
II tRNA consists of variable arm. 13-20 bp
Tertiary structure
Has 3D structure that is highly folded & has an L-shaped structure.
On the 3° structure of tRNA acceptor & TUC arm form the extended helix downwards. The
anticodon & DHU arm form the extended helix upwards. Due to change in structured
configuration the DHU & TUC join where both the extended helices align at an angle of 90°.
Function
The stabilization of 3° structure is through the base pairing & base stacking
Function
tRNA first recognizes the codons of mRNA with the help of anticodon arm. The anticodon
arm of tRNA consists of 3 anticodons, which when Complementarily bands with Codons of
mRNA. That which after binding activates the synthesis of an enzyme known as aminoacyl
tRNA synthetase.
The synthetase enzyme will help in the binding of specific an according to the mRNA
specific codons that will code.
The particular aa when both aa & tRNA molecules attaches, with synthetase enzyme, it will
promote both t-RNA and aa bonding day utilizing an ATP molecule. Once the synthetase
enzyme charges the t-RNA, it will load the aa and transfer it to ribosome where the aa
combines to build up of protein
R-rna – Ribosome RNA –
The ribosomes are the factories of protein synthes. The eukaryotic ribosomes are
composed of two major nucleoprotein complexes 60S subunit & 40S subunit. The 60S
contains 28S rRNA, 5S rRNA and 5.8S rRNA while 40S contains 18S RNA. Prokaryotic
ribosomes are dense structures which are 50S and 30S type. 50S and 30S are large & small
subunits of bac. Having 70S type of ribos. ‘S’ denotes the Svedberg unit which measures
the sedimentation coefficient of ribosome in an ultracentrifuge.
Ribosome associated with cytoplasmic membrane → prokaryotes
Ribosome + ER in eukaryotes.
Prokaryots = 70S type = 30S (small) + 50S (lLarge nucleoproteins particles containing RNA)
When 2 Subunits combine their
sedimentation rate is not additive becoz
the shape & mass change, leading to a
combined rate is less than the sum of
proteins of individual units.
30s = 16 s + 21 protein. , 50 s= 5s + 23 s + 31 protein
Both have 3 sites for aassociation of trna
Molecular weight 2.4X 106 Daltons
30S bind mRna ,50s bind 30s form complete 70 for protein synthesis unit
A = denotes aminoacyl chain, accepts the incoming amino acylated tRNA.
P = peptidyl chain. It holds the tRNA with nascent peptide chain.
E = exit site. It holds the deacylated tRNA
Function
30S: provide binding site for incoming transcribed [Link] the small subunit of
prokaryotic ribosome contains specific base-pairing between the codon and anticodon on
the mRNA and tRNA respectively.
50S (larger subunit of prokaryote) mediates the peptide bond formation during the peptidyl
transfer reaction. It also acts as the site of inhibition for many antibiotics & prevents
premature polypeptide hydrolysis & helps in protein folding after translation.
Base pairing – The two antiparallel DNA polynucleotide strands that make up the DNA
molecules are held together by hydrogen bonds between the nitrogenous bases. These H-
bonds always occur between the same pairs of bases.
The purine Adenine always pairs with pyrimidine Thymine with 2 hydrogen bonds formed
between those bases.
The purine Guanine always pairs with pyrimidine Cytosine with 3 hydrogen bonds formed
between those bases.
This is known as Complementary base pairing.
These pairs were known as DNA base pairs.
Complementary Base Pairing-
Complementary base pairing is defined as the phenomenon wherein the guanine always
hydrogen bonds to the cytosine and adenine binds to thymine always.
The Watson crick base pairing, which is the foundation for the helical structure of DNA, &
intramolecular base pairs can take place within the single stranded nucleic acid.
Particularly, this is essential in RNA molecules (+-RNA) where the watson crick base pairs
(A-U & G-C) permit the formation of short double strand helices. Additionally, the base-
pairing between the mRNA and tRNA forms the basis for the molecular recognition events,
which result in the nucleotide sequence of the mRNA becoming translated into the
sequence of proteins through the genetic code. Often, the size of either an entire genome
or an individual gene of an organism is measured in the base pair, because usually DNA is
ds. Thus the total BP counts =nucleotides count in any one of the strands.
The haploid human genome (23 chromosomes) can also estimated to be nearly 3.2 B bases
long & have 20K – 25K distinct protein-coding genes.
Hydrogen Bonding and Stability
Hydrogen bonding is given as the chemical interaction which underlies the base pairing
rule. Only the right pairs will produce stability due to an effective geometrical
correspondence of both hydrogen bond donors and acceptors. DNA pairs having high
GC content is stable compared to the DNA with low GC content. But the hydrogen bonds
do not significantly stabilize the DNA, stabilization is primarily because of stacking
interactions.
The bigger nucleobases guanine and adenine are the members of the double-ringed
chemical structures class known as Purines; the smaller nucleobases thymine and
cytosine (including uracil), are the member of a single-ringed chemical structures, class
known as Pyrimidines.
And Purines are only complementary with the Pyrimidines. Pairing of pyrimidine-
pyrimidine are energetically unfavourable due to molecules are too far apart for
hydrogen bonding that is to be established; Purine-purine pairing are energetically
unfavourable since the molecules are too close together, resulting in overlap repulsion.
Hypochromotacity & Hyperchromaticity of DNA
Hyperchromaticity –
It is the increase of absorbance (optical density) of a material. The most famous example is
the hyperchromaticity of DNA that occurs when the DNA duplex is denatured. The UV
absorption is increased when the two single DNA strands are being separated either by
heat or by addition of denaturant or by increasing the pH.
→ Heat denaturation of DNA, also called melting, causes the double helix structure to
unwind to Form single stranded DNA, when DNA in solution is heated above its melting
temp (Tm) all the ds-DNA unwinds to form ss-DNA.
The bases become unstacked and can thus absorb more light.
In their native state the bases of DNA absorb light in the 260 nm wavelength region when
the chains become unstacked the wavelength of max absorbance does not change, but the
amount absorbed increases by 37%
A ds-DNA strand dissociating to two ss-strands produces a sharp cooperative transition.
Hyperchromicity can be used to track the condition of DNA as temp changes. The
transition/melting temp (Tm) is the temp where the absorbance of UV light is \(50\%\) down
the max abs, i.e. where 50% of the DNA is denatured. A ten fold increase of monovalent
cation conc increased the temp by 16.6^
The hyperchromic effect is the striking increase in the absorbance of DNA upon
denaturation. The two strands of DNA are bound together mainly by stacking interaction,
hydrogen bonds & hydrophobic effect between the complementary bases. The H bond
limits the resonance of the aromatic ring so the absorbance of the base pairs is limited as
well.
Hypochromicity
Hypochromicity refers to a material’s electronics ability to absorb light. The hypochromic
effect describes the decrease in the absorbance of uv light in a ds DNA compared to its ss
DNA counter part. Compared to a ss DNA, a ds DNA consists of stacked bases that
contribute to the stability & the hypochromicity of the DNA.
When a double stranded DNA is denatured, the stacked bases break apart & thus become
less stable. It also absorbs more ultraviolet light since the bases no longer form hydrogen
bonds & there force vaves free to absorb light. Ways to denature DNA include high
temperature addition & increasing the pH level.
Importance –
The measurement of absorption of light is important in monitoring the melting & annealing
of DNA.
At the melting temp (Tm), the DNA is half denatured & half ds...
By lowering the temp below the Tm, the denatured DNA strands would anneal back into a
ds-DNA, when temp is above the Tm, the DNA is denatured.
Because melting occurs almost instantly at a certain temp, monitoring the absorbance of
the DNA at various temp would indicate the Tm.
By being able to find the temp at which DNA melted and annealed, scientists are able to
separate DNA strands & anneal them back with other DNA strands
This is important in creating hybrid DNA which consists of two DNA strands from different
sources.
Since DNA strands can only anneal if they are similar, the creation of hybrid DNA can
indicate similarities b/w genomes of different organisms.