Chemistry

Lecture 22/ Nucleic Acids 6

DNA Transcription : [RNA Structure, Synthesis &Processing]

In order that the genetic map is expressed into the phenotypic features, parts of the DNA known
as ‘GENES” have to be expressed.
Gene expression involves TWO things:
❖ DNA transcription (production of RNAs- the working copies of DNA)
❖ RNA translation (leads to proteins production)
 Structure of RNAs
There are three major types of RNA that all participate in the process of protein synthesis:
❖ ribosomal RNA (rRNA),
❖ transfer RNA (tRNA),
❖ messenger RNA (mRNA).
These are unbranched polymeric chains composed of ribonucleoside monophosphates joined
together by phosphodiester bonds [their pentose sugar moiety is ribose].
A. Ribosomal RNA (r RNA)
rRNAs are present in association with several proteins as structural components of the
ribosomes.
Together, rRNA make up about 80% of the total
RNA in the cell and some rRNA function as
catalysts in protein synthesis.
Note: “S” is the Svedberg unit, which is related to
the molecular weight and shape of the
compound], also its Sedimentation coefficient.
B. Transfer RNA
➢ t-RNA(s) are the smallest (4S) of the three major species of RNA.
➢ Their job is to transfer amino acids from their pool in the cytoplasm to ribosomes.
➢ There is at least one specific t-RNA molecule for each of the 20 amino acids commonly found
in proteins.
➢ Together, t-RNA make up about 15% of the total RNA in the cell.
N.B.: Some amino acids are transferred by more than one type of t-RNA.
There are 2 important features of t-RNA:
1. They contain unusual bases e.g, dihydrouracil (D) and pseudouracil (ψ) .
2. They have extensive complementary intrachain base-pairing leading to extensive looping and
folding giving rise to the secondary and tertiary structures of t-RNA..

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Each t-RNA can recognize the signal (message) on the mRNA

(codon sequences) by its anticodon loop, so that it brings up the
specific amino acid covalently attached to its 3′-end to add it to
the growing polypeptide chain during protein synthesis.
C. Messenger RNA (mRNA)
It comprises only about 5% of the RNA in the cell. It carries the
message (i.e. the genetic information) from the nuclear DNA to
the cytosol, where it is used as the template for protein synthesis.
It is widely different in size and base sequences. The mature
mRNA contains:
❖ coding regions to be translated to proteins later on in addition
to
❖ untranslated regulatory regions (UTR) at both 5′- and 3′-ends.
Special structural characteristics of the mature eukaryotic
mRNA include:
✓ a long sequence of adenine nucleotides (a “poly-A tail”) on the
3′-end of the RNA chain
✓ a “cap” on the 5′-end consisting of a molecule of 7-methylguanosine attached “backward”
(5′→5′) through a triphosphate linkage.

The structure of
mature mRNA.

 Transcription of Eukaryotic Genes

The transcription of eukaryotic genes is a far more complicated process than transcription in
prokaryotes.
There are specific separate polymerases for the synthesis of different species of RNA (rRNA,
tRNA, and mRNA).
In addition, a number of proteins called transcription factors are involved. These factors have
two main functions:
1. They recognize which genes are to be transcribed now?
2. They help in the assembly of the components of transcription complex at the promoter
region of that gene.
[Note: Each eukaryotic RNA polymerase has its own promoters and transcription factors].

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 Chromatin Remodeling [DNA Opening-Up]

For transcription factors to recognize and bind to their specific DNA sequences, the chromatin
structure in that region must be altered to allow access to the DNA.
A major mechanism by which chromatin is remodeled is through acetylation of lysine residues
at the amino terminus of histone proteins.
This process of opening up the chromatin and making DNA more accessible for transcription is
mediated by histone acetyltransferases.
Removal of the acetyl group by histone deacetylases restores the positive charge of histones,
and makes stronger interactions between histones and DNA again.
 RNA Polymerases
There are 3 classes of RNA polymerase in the nucleus of eukaryotic cells. All are large enzymes
with multiple subunits. Each class of RNA polymerase recognizes particular types of genes:
RNA polymerase I: It synthesizes the precursor of the 28S, 18S, and 5.8S r-RNA in the nucleolus.
RNA polymerase II: It synthesizes the precursors of m-RNA in addition to most of snRNA species.
RNA polymerase III: It produces the small RNA types, including t-RNA, 5S ribosomal RNA, and
some snRNA.
Steps of RNA Synthesis (DNA Transcription)
1. Initiation: begins with binding of RNA polymerase to a region in the DNA known as promoter.
In eukaryotic cells, each class of RNA polymerase has specific transcription factors and specific
promoter areas.
When a DNA sequence in a given gene is to be transcribed to mRNA using RNA polymerase II,
the ‘promoter’ consensus sequence is called TATA or Hogness box that is centered about 25
nucleotides to the left of the transcription start site.
Another consensus sequence is found between 70 and 80 nucleotides to the left as well and
called CAAT box.

In some eukaryotic genes, TATA box is substituted by GC-rich region (GC box).
Such sequences in the promoter area serve as binding sites for proteins known as transcription
factors, which in turn interact with each other and with RNA polymerase II.

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Eukaryotic RNA polymerase II does not itself recognize and bind the promoter, but is brought to
the promoter area by transcription factors (mainly a general transcription factor, TFIIF).

In addition to binding DNA, specific transcription factors also bind other proteins (“co-
activators”), recruiting them to the transcription complex.
[Note: Co-activators may include enzymes like histone acetyl transferase which is involved in
chromatin remodeling]
In order to enhance this step (initiation), there are probably thousands of specific DNA
sequences away from the transcription start site (toward both 5′- and 3′-ends) called as
Enhancers or Response Elements (RE) that bind to specific proteins called Activators which
interact with the “Transcription complex” to stimulate the transcription.
N.B. Enhancers (RE)& their Activators are not part of “Transcription Complex” as they are not
present in the promoter area, but interact with it.
2. Elongation:
Once the promoter region has been recognized and bound by the transcription complex, local
unwinding (melting) of DNA helix occurs.
RNA polymerase II begins to synthesize a transcript of the DNA sequence of one of the strands.
The elongation phase is said to begin when:
1. The transcript (typically starting with a purine) exceeds 10 nucleotides in length
2. RNA polymerase starts to leave the promoter region.
During transcription process, a short temporary “DNA-RNA hybrid helix” is formed.

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3. Termination:
The elongation of the single-stranded RNA chain continues until a termination signal is reached.
Termination can be intrinsic (spontaneous) or dependent upon a protein known as ρ (rho) factor.
This leads to stop transcription, separation of the primary transcript and the DNA double helix
to zip up again (role of histone de-acetylase).
 Post-transcriptional Modification of RNAs
The RNA strand that has been produced from DNA transcription
is known as the “primary transcript”.
The primary transcript of rRNA and tRNA are transcriptionally
modified by cleavage of the original transcript by ribonucleases.
tRNA will be further modified to give each species its unique
identity.
mRNA primary transcript will be extensively modified as well.
r-RNA of both prokaryotic and eukaryotic cells are synthesized
from long precursor molecules called (pre-ribosomal RNAs).
The 28S, 18S, and 5.8S rRNA of eukaryotes are produced by the cleavage of the primary
transcript by ribonuclease enzyme.
Transfer RNA posttranscriptional modification
Functional t-RNA is produced from a longer precursor molecule (pre- t RNA) after modifications
which include:
1. An intron (14 nucleotides) must be removed from the anticodon loop
2. A sequences at both the 5′- and the 3′-ends of the molecule must be trimmed out.
3. addition of CCA sequence by nucleotidyltransferase to the 3′-terminal end of tRNA
4. Modification of bases at specific positions to produce “unusual bases” like (D=
Dihydrouracil, ψ= pseudouracil, and m= methylated bases).

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Eukaryotic mRNA posttranscriptional modification

The mRNA molecule synthesized in eukaryotic nuclei by RNA polymerase II is a collection of the
precursor molecules of mRNA called as heterogeneous nuclear RNA (hnRNA). The primary
transcripts are extensively modified in the nucleus after transcription. These modifications
usually include:
1. 5′ “Capping”: This process is the first of the processing reactions for hnRNA. The cap is a [7-
methylguanosine triphosphate] attached “backward” to the 5′-terminal end of the mRNA,
forming an unusual 5′→5′ triphosphate linkage.

The presence of this 7- methyl guanosine triphosphate cap is very essential in starting the mRNA
translation later on (i.e. protein synthesis).
2. Addition of a poly-A tail
Most eukaryotic mRNA have a chain of 40–200 adenine nucleotides attached to the 3′-end of
mRNA primary transcript.
This poly-A tail is not transcribed from the DNA, but added after DNA transcription by the
nuclear enzyme, polyadenylate polymerase, using ATP as the substrate.
These tails help stabilize the mRNA and facilitate their exit from the nucleus. After the mRNA
enters the cytosol, the poly-A tail is gradually shortened.
3. Removal of introns
Maturation of eukaryotic mRNA usually involves the removal of RNA sequences, which do not
code for protein (introns, or intervening sequences) from the primary transcript.
The remaining coding sequences, the exons, are joined together to form the mature mRNA.
The process of removing introns and joining exons is called splicing. The molecular machine that
accomplishes these tasks is known as the spliceosome.

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