DNA transcription & Post Transcriptional Modification

Complete notes on DNA transcription & Post Transcriptional Modification.

1. DNA Transcription
&
Post Transcriptional Modification
Hafiz.M.Zeeshan.Raza
Research Assistant_HEC_NRPU
[email protected]
COMSATS UNIVERSITY SAHIWAL

3. Transcription in Prokaryotes and Eukaryotes

4. RNA
Ribonucleic acid is a polymeric molecule essential in various
biological roles in coding, decoding, regulation, and expression of
genes.
Polymer of ribonucleotide held together by 3’ to 5’ phosphodiester
bridge and are single stranded.
RNA is the only molecule known to function both in the storage and
transmission of genetic information and in catalysis.
All RNAs except the RNA genomes of certain viruses derive
information that is permanently stored in DNA.

5. Kinds of RNA
There are 4 types of RNA, each encoded
by its own type of gene.
The genomic DNA contains all the
information for the structure and function
of an organism.
In any cell, only some of the genes are
expressed, that is, transcribed into RNA.

6. Continue…
mRNA - Messenger RNA: Encodes amino acid sequence of a
polypeptide.
tRNA - Transfer RNA: Brings amino acids to ribosomes during
translation.
rRNA - Ribosomal RNA: With ribosomal proteins, makes up the
ribosomes, the organelles that translate the mRNA.
snRNA - Small nuclear RNA: With proteins, forms complexes
that are used in RNA processing in eukaryotes. (Not found in
prokaryotes.)

7. Features of transcription
1. Highly Selective –
This selectivity is due to signals embedded in the nucleotide sequence of DNA.
Specific sequences mark the beginning and the end of DNA segment which is o
be transcribed.
This signal instruct the enzyme where to start and stop transcription, when to start
and how often to start.
2. Formation of Primary Transcripts –
Many of RNA transcripts are synthesized as precursors known as Primary
transcripts.
On modification and trimming they are converted into functional RNA.

8. RNA polymerase
RNA polymerases are enzymes that transcribe DNA into RNA.
Using a DNA template, RNA polymerase builds a new RNA molecule through base
pairing.
For instance, if there is a G in the DNA template, RNA polymerase will add a C to the
new, growing RNA strand.
RNA polymerase always builds a new RNA strand in the 5’ to 3’ direction. That is, it
can only add RNA nucleotides (A, U, C, or G) to the 3' end of the strand.
RNA polymerases are large enzymes with multiple subunits, even in simple
organisms like bacteria.
In addition, humans and other eukaryotes have three different kinds of RNA
polymerases: I, II, and III. Each one specializes in transcribing certain classes of
genes.

9. Transcription initiation
To begin transcribing a gene, RNA polymerase binds to the DNA of the
gene at a region called the promoter.
Basically, the promoter tells the polymerase where to "sit down" on the DNA
and begin transcribing.
Each gene (or, in bacteria, each group of genes transcribed together) has
its own promoter.
A promoter contains DNA sequences that let RNA polymerase or its helper
proteins attach to the DNA.
Once the transcription bubble has formed, the polymerase can start
transcribing.

11. Promoters in bacteria
To get a better sense of how a promoter works, let's look an example from
bacteria.
A typical bacterial promoter contains two important DNA sequences, the -10 and
- 35 elements.
RNA polymerase recognizes and binds directly to these sequences.
The sequences position the polymerase in the right spot to start transcribing a
target gene, and they also make sure it's pointing in the right direction.
Once the RNA polymerase has bound, it can open up the DNA and get to work.
DNA opening occurs at the -10 element, where the strands are easy to separate
due to the many As and Ts (which bind to each other using just two hydrogen
bonds, rather than the three hydrogen bonds of Gs and Cs).

13. Promoters in humans
In eukaryotes like humans, the main RNA polymerase in your cells does not
attach directly to promoters like bacterial RNA polymerase.
Instead, helper proteins called basal (general) transcription factors bind to
the promoter first, helping the RNA polymerase in your cells get a foothold
on the DNA.
Many eukaryotic promoters have a sequence called a TATA box.
It's recognized by one of the general transcription factors, allowing other
transcription factors and eventually RNA polymerase to bind.
It also contains lots of As and Ts, which make it easy to pull the strands of
DNA apart.

15. Elongation
Once RNA polymerase is in position at the promoter, the next step
of transcription—elongation—can begin.
Basically, elongation is the stage when the RNA strand gets longer,
thanks to the addition of new nucleotides.
During elongation, RNA polymerase "walks" along one strand of
DNA, known as the template strand, in the 3' to 5' direction.
For each nucleotide in the template, RNA polymerase adds a
matching (complementary) RNA nucleotide to the 3' end of the RNA
strand.

16. Continue…
The RNA transcript is nearly identical to the non-template, or coding, strand of DNA.
However, RNA strands have the base uracil (U) in place of thymine (T), as well as a
slightly different sugar in the nucleotide.
So, as in the diagram , each T of the coding strand is replaced with a U in the RNA
transcript.

17. Termination
RNA polymerase will keep transcribing until it gets
signals to stop.
The process of ending transcription is called termination,
and it happens once the polymerase transcribes a
sequence of DNA known as a terminator.

18. Termination in bacteria
There are two major termination strategies found in bacteria: Rho-dependent and
Rho- independent.
In Rho-dependent termination, the RNA contains a binding site for a protein called
Rho factor. Rho factor binds to this sequence and starts "climbing" up the transcript
towards RNA polymerase.
When it catches up with the polymerase at the transcription bubble, Rho pulls the
RNA transcript and the template DNA strand apart, releasing the RNA molecule and
ending transcription.
Another sequence found later in the DNA, called the transcription stop point, causes
RNA polymerase to pause and thus helps Rho catch up.
Rho-independent termination depends on specific sequences in the DNA template
strand.

19. Continue…
As the RNA polymerase approaches the end of the gene being transcribed,
it hits a region rich in C and G nucleotides.
The RNA transcribed from this region folds back on itself, and the
complementary C and G nucleotides bind together.
The result is a stable hairpin that causes the polymerase to stall.
 In a terminator, the hairpin is followed by a stretch of U nucleotides in the
RNA, which match up with A nucleotides in the template DNA.
The complementary U-A region of the RNA transcript forms only a weak
interaction with the template DNA.
This, coupled with the stalled polymerase, produces enough instability for
the enzyme to fall off and liberate the new RNA transcript.

21. Key points
Transcription is the process in which a gene's DNA sequence is copied
(transcribed) to make an RNA molecule.
RNA polymerase is the main transcription enzyme.
Transcription begins when RNA polymerase binds to a promoter sequence
near the beginning of a gene (directly or through helper proteins).
RNA polymerase uses one of the DNA strands (the template strand) as a
template to make a new, complementary RNA molecule.
Transcription ends in a process called termination. Termination depends
on sequences in the RNA, which signal that the transcript is finished.

22. Post transcriptional modification
The primary transcript needs to be modified to become functional
tRNAs, rRNAs and mRNAs.
Post Transcriptional modifications include;
Splicing
Addition of 5’ cap
Creation of Poly A tail
RNA editing.

23. Pre-mRNA processing (splicing)
Eukaryotic pre-mRNAs typically include introns.
Introns are removed by RNA processing in which the intron is looped
out and cut away from the exons by snRNPs, and the exons are
spliced together to produce the translatable mRNA.
The steps of pre-mRNA splicing (intron removal) are as follows:
The intron loops out as snRNPs (small nuclear ribonucleoprotein particles,
complexes of snRNAs and proteins) bind to form the spliceosome.
The intron is excised, and the exons are then spliced together.
The resulting mature mRNA may then exit the nucleus and be translated in
the cytoplasm.

25. Addition of 5’ Cap
 At the end of transcription, the 5' end of the RNA transcript contains
a free triphosphate group since it was the first incorporated
nucleotide in the chain.
The capping process replaces the triphosphate group with another
structure called the "cap".
The cap is added by the enzyme guanyl transferase.
This enzyme catalyzes the reaction between the 5' end of the RNA
transcript and a guanine triphosphate (GTP) molecule.

26. The poly A Tail
Post-transcriptional RNA processing at the opposite end of the transcript
comes in the form of a string of adenine bases attached to the end of the
synthesized RNA chain.
This string of adenine is called the "poly A tail".
The addition of the adenines is catalyzed by the enzyme poly (A)
polymerase, which recognizes the sequence AAUAAA as a signal for the
addition.
The reaction proceeds through mechanism similar to that used for the
addition of nucleotides during transcription.
The poly A tail is found on most, but not all, eukaryotic RNA transcripts. Its
significance remains unknown.

28. RNA editing
RNA (ribonucleic acid) editing is a post-transcriptional alteration of RNA
sequences and structures via modification, deletion or insertion of
nucleotides.
This process has been detected in eukaryotes ranging from single-celled
protozoa to plants and mammals; as a result, functionally distinctive proteins
can be processed from a single gene.
RNA editing occurs concurrently with transcription and splicing processes in
the nucleus or mitochondria.
It comes in a myriad of different natures, with all of the diverse editing types
distributed intermittently across the phylogenetic spectrum.