This document discusses post-transcriptional modifications of RNA and inhibitors of transcription. It describes how primary transcripts of rRNA, tRNA and mRNA undergo processing in both prokaryotes and eukaryotes. For rRNA, introns are removed and exons are ligated. For tRNA, extra nucleotides are removed, bases are modified and the CCA tail is added. For mRNA, a 5' cap is added, introns are spliced out, a poly-A tail is attached. Splicing produces tissue-specific proteins. Inhibitors like rifampicin, actinomycin D and alpha-amanitin block transcription by binding DNA or RNA polymerase.
it describes transcription with simple diagram and animation. its steps and inhibitors are described for both eukaryotes and prokaryotes. it will be easily understood by UG students . post transcriptional modification of all the RNA are also described with diagrams.
it describes transcription with simple diagram and animation. its steps and inhibitors are described for both eukaryotes and prokaryotes. it will be easily understood by UG students . post transcriptional modification of all the RNA are also described with diagrams.
RNA Polymerase
Introduction
Purification
History
PRODUCTS OF RNAP
Messenger RNA
Non-coding RNA or "RNA genes
Transfer RNA
Ribosomal RNA
Micro RNA
Catalytic RNA (Ribozyme)
prokaryotic and eukaryotic
Transcription by RNA Polymerase
TYPES OF RNA POLYMERASE
Type I
Type II
Type III
Prokaryotic Transcription Unit
EXPRESSION OF A PROKARYOTIC GENE
Prokaryotic Polycistronic Message Codes for Several Different Proteins
Eukaryotic Transcription Unit
ENHANCERS AND SILENCERS
RESULT OF THE TRANSCRIPTION CYCLE
RNAP III TRANSCRIBES HUMAN MICRORNAS
RNAP I–specific subunits promotepolymerase clustering to enhance the rRNA genetranscription cycle
RNAP II–TFIIB STRUCTURE ANDMECHANISM OF TRANSCRIPTION INITIATION
FIVE CHECKPOINTS MAINTAINING THE FIDELITY OFTRANSCRIPTION BY RNAP IN STRUCTURAL ANDENERGETIC DETAILS
This presentation is about the transcription machinery that is required for the transcription in eukaryotes. The comparison between the transcription factors involved in prokaryotes and eukaryotes. The initiation of transcription and how it helps in producing a mRNA.
Eukaryotic transcription is the elaborate process that eukaryotic cells use to copy genetic information stored in DNA into units of RNA replica.- Source: Wikipedia
RNA Polymerase
Introduction
Purification
History
PRODUCTS OF RNAP
Messenger RNA
Non-coding RNA or "RNA genes
Transfer RNA
Ribosomal RNA
Micro RNA
Catalytic RNA (Ribozyme)
prokaryotic and eukaryotic
Transcription by RNA Polymerase
TYPES OF RNA POLYMERASE
Type I
Type II
Type III
Prokaryotic Transcription Unit
EXPRESSION OF A PROKARYOTIC GENE
Prokaryotic Polycistronic Message Codes for Several Different Proteins
Eukaryotic Transcription Unit
ENHANCERS AND SILENCERS
RESULT OF THE TRANSCRIPTION CYCLE
RNAP III TRANSCRIBES HUMAN MICRORNAS
RNAP I–specific subunits promotepolymerase clustering to enhance the rRNA genetranscription cycle
RNAP II–TFIIB STRUCTURE ANDMECHANISM OF TRANSCRIPTION INITIATION
FIVE CHECKPOINTS MAINTAINING THE FIDELITY OFTRANSCRIPTION BY RNAP IN STRUCTURAL ANDENERGETIC DETAILS
This presentation is about the transcription machinery that is required for the transcription in eukaryotes. The comparison between the transcription factors involved in prokaryotes and eukaryotes. The initiation of transcription and how it helps in producing a mRNA.
Eukaryotic transcription is the elaborate process that eukaryotic cells use to copy genetic information stored in DNA into units of RNA replica.- Source: Wikipedia
RNA- A polymer of ribonucleotides, is a single stranded structure. There are three major types of RNA- m RNA,t RNA and r RNA. Besides that there are small nuclear,micro RNAs, small interfering and heterogeneous RNAs. Each of them has a specific structure and performs a specific function.
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Gene therapy is an experimental treatment that involves introducing genetic material into a person’s cells to fight or prevent disease. Researchers are studying gene therapy for a number of diseases, such as severe combined immuno-deficiencies, hemophilia, Parkinson's disease, cancer and even HIV, through a number of different approaches (see video: 'Gene Therapy a new tool to cure human diseases'). A gene can be delivered to a cell using a carrier known as a “vector.” The most common types of vectors used in gene therapy are viruses. The viruses used in gene therapy are altered to make them safe, although some risks still exist with gene therapy. The technology is still in its infancy, but it has been used with some success.
Post-transcriptional modification or co-transcriptional modification is a set of biological processes common to most eukaryotic cells by which an RNA primary transcript is chemically altered following transcription from a gene to produce a mature, functional RNA molecule
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2. Contents
Post Transcriptional Modifications of-
• Ribosomal RNA
• Transfer RNA
• Messenger RNA
Clinical significance of Splicing
Biological Significance of Splicing
Inhibitors of Transcription
2Biochemistry For Medics- Lecture Notes
3. Primary Transcript
• A primary transcript is a linear copy of a
transcriptional unit-The segment of DNA
between specific initiation and termination
sequences.
3Biochemistry For Medics- Lecture Notes
4. Modifications of prokaryotic
primary transcript
• The primary transcript of prokaryotic
• t- RNA s and r -RNA s are post-
transcriptionally modified by
removing extra nucleotides .
• t RNA s are then further modified to acquire
special characters unique to t- RNAs.
Biochemistry For Medics- Lecture Notes 4
5. Modifications of primary transcript in
prokaryotes
• In prokaryotic organisms, the primary
transcripts of mRNA-encoding genes begin to
serve as translation templates even before
their transcription has been completed.
• This is because the site of transcription is not
compartmentalized into a nucleus as it is in
eukaryotic organisms.
• Consequently, prokaryotic mRNAs are
subjected to little processing prior to carrying
out their intended function in protein
synthesis.
5Biochemistry For Medics- Lecture Notes
6. Modifications of primary
transcript of mRNA in prokaryotes
• Transcription and translation are coupled in
prokaryotic cells.Biochemistry For Medics- Lecture Notes 6
7. Post Transcriptional modifications in
Eukaryotes
• All eukaryotic RNA primary transcripts undergo
extensive processing whether it be as mRNA or as a
component of the translation machinery such as
rRNA, 5S RNA, or tRNA or RNA processing
machinery, snRNAs.
• Processing occurs primarily within the nucleus and
includes nucleolytic cleavage to smaller molecules
and coupled nucleolytic and ligation reactions
(splicing of exons).
• The processes of transcription, RNA processing,
and even RNA transport from the nucleus are
highly coordinated.
7Biochemistry For Medics- Lecture Notes
8. Processes involved in the post
transcriptional modifications
• Some of the processes involved in the post
transcriptional modifications of primary
transcript of major RNAs are as follows-
A) Ribosomal RNA
• In mammalian cells, the three rRNA
molecules are transcribed as part of a single
large precursor molecules called , Pre
ribosomal RNAs.
8Biochemistry For Medics- Lecture Notes
9. Post Transcriptional modifications of
Ribosomal RNA(r- RNA)
• The precursor is subsequently processed in
the nucleolus to provide the RNA component
for the ribosome subunits found in the
cytoplasm.
• The 23S,16S, and 5S ribosomal RNAs of
prokaryotes are produced form a single RNA
precursor molecule as are the 28S, 18S and
5.8S r RNAs of eukaryotes.
• Eukaryotic 5S rRNA in eukaryotes is
synthesized by RNA polymerase III and
modified separately.
9Biochemistry For Medics- Lecture Notes
10. Post Transcriptional modifications of
Ribosomal RNA (r- RNA)
• The 23S,16S, and 5S ribosomal RNAs of prokaryotes are
produced form a single RNA precursor molecule
• Cleavage and trimming are the mechanisms involved,
• Similar modifications are observed in the processing of
eukaryotic r-RNA.
10Biochemistry For Medics- Lecture Notes
11. Post Transcriptional modifications of
Ribosomal RNA(r- RNA)
• 28S, 18S and 5.8S r RNAs are synthesized from a
single 45 S precursor in eukaryotes.
• Eukaryotic 5S rRNA is synthesized by RNA
polymerase III and modified separately.
Biochemistry For Medics- Lecture Notes 11
12. Post Transcriptional modifications of
Transfer RNA (t- RNA)
• The tRNA molecules serve as adapter
molecules for the translation of mRNA into
protein sequences.
• Both eukaryotic and prokaryotic transfer
RNA s are made from longer precursor
molecules that must be modified.
• The basic mechanism involved are as
follows-
12Biochemistry For Medics- Lecture Notes
13. Post Transcriptional modifications of
Transfer RNA(t- RNA)
• Splicing- An intron must be removed from the
anticodon loop
• Trimming- The sequences at both the 5' and
3' ends of the molecule are trimmed
• Base modifications-The tRNAs contain many
modifications of the standard bases A, U, G, and
C, including methylation, reduction,
deamination, and rearranged glycosidic bonds.
Further modification of the tRNA molecules
includes nucleotide alkylations,
13Biochemistry For Medics- Lecture Notes
14. Post Transcriptional modifications of
Transfer RNA(t- RNA)
• CCA attachment
• The attachment of the characteristic CpCpAOH
terminal at the 3' end of the molecule by the
enzyme nucleotidyl transferase is the most
important modification.
• The 3' OH of the A ribose is the point of
attachment for the specific amino acid that is to
enter into the polymerization reaction of
protein synthesis
14Biochemistry For Medics- Lecture Notes
15. Post Transcriptional modifications of
Transfer RNA(t- RNA)
The extra
nucleotides at both
5' and 3' ends of t-
RNA are removed,
an intron from the
anticodon arm is
removed, bases are
modified and CCA
arm is attached to
form the mature
functional t RNA.
15Biochemistry For Medics- Lecture Notes
16. Post Transcriptional modifications of
pre m- RNA
• In prokaryotic organisms, the primary
transcripts of mRNA-encoding genes begin
to serve as translation templates even
before their transcription has been
completed.
• In all eukaryotes the primary transcripts of
mRNA-encoding genes undergo extensive
processing before they are converted to
mature functional forms
16Biochemistry For Medics- Lecture Notes
17. Post Transcriptional modifications of
pre m- RNA
a) 5' Capping
• Mammalian mRNA molecules contain a 7-
methylguanosine cap structure at their 5' terminal.
• The cap structure is added to the 5' end of the newly
transcribed mRNA precursor in the nucleus prior to
transport of the mRNA molecule to the cytoplasm.
• The 5' cap of the RNA transcript is required both for
efficient translation initiation and protection of the
5' end of mRNA from attack by 5-'3' exonucleases.
• Eukaryotic m RNAs lacking the cap are not
efficiently translated.
17Biochemistry For Medics- Lecture Notes
18. Post Transcriptional modifications of
pre m- RNA
•The addition of the
Guanosine triphosphate
(part of the cap is
catalyzed by the nuclear
enzyme guanylyl
transferase.
•Methylation of the
terminal guanine occurs
in the cytoplasm. and is
catalyzed by guanine-7-
methyl transferase.
•S-Adenosyl methionine is the methyl group
donor.
•Additional methylation steps may occur.
The secondary methylations of mRNA
molecules, those on the 2'-hydroxy and the
•N6 of adenylyl residues, occur after the mRNA
molecule has appeared in the cytoplasm
18Biochemistry For Medics- Lecture Notes
19. Post Transcriptional modifications of
pre m- RNA
b) Addition of poly A tail
• Poly(A) tails are added to the 3' end of mRNA molecules in a
posttranscriptional processing step.
• The mRNA is first cleaved about 20 nucleotides downstream
from an AAUAA recognition sequence
• Another enzyme, poly(A) polymerase, adds a poly(A) tail which
is subsequently extended to as many as 200 A residues.
• The poly(A) tail appears to protect the 3' end of mRNA from 3'
5' exonuclease attack.
• Histone and interferon's mRNAs lack poly A tail.
• After the m-RNA enters the cytosol, the poly A tail is gradually
shortened.
19Biochemistry For Medics- Lecture Notes
21. Post Transcriptional modifications of
Pre m RNA
Removal of introns (Splicing)
• Introns or intervening sequences are the RNA
sequences which do not code for the proteins.
• These introns are removed from the primary
transcript in the nucleus, exons (coding
sequences) are ligated to form the mRNA
molecule, and the mRNA molecule is
transported to the cytoplasm.
• The steps of splicing are as follows-
21Biochemistry For Medics- Lecture Notes
22. Post Transcriptional modifications of
Pre m RNA
• Introns are removed from the primary transcript
in the nucleus, exons (coding sequences) are
ligated to form the mRNA molecule
Biochemistry For Medics- Lecture Notes 22
23. Splicing of m-RNA
The steps of splicing are as follows-
• i) Role of small nuclear RNA (sn RNA) and
Spliceosome
• The molecular machine that accomplishes the task
of splicing is known as the spliceosome.
Spliceosomes consist of the primary transcript, five
small nuclear RNAs (U1, U2, U4, U5, and U6) and
more than 60 proteins.
• Collectively, these form a small ribonucleoprotein
(snRNP) complex, sometimes called a "snurp"
(snRNPs)
23Biochemistry For Medics- Lecture Notes
24. Splicing of m-RNA
• Snurps are thought to position the RNA
segments for the necessary splicing
reactions.
• These facilitate the splicing of exon
segments by forming base pairs with the
consensus sequence at each end of the
intron.
24Biochemistry For Medics- Lecture Notes
25. Splicing consensus Sequences
There are reasonably conserved sequences at
each of the two exon-intron (splice) junctions
Biochemistry For Medics- Lecture Notes 25
27. Splicing of m-RNA
ii) Mechanism of excision of introns
• The binding of snRNPs brings the sequences
of the neighboring exons in to the correct
alignment for splicing.
• The 2'-OH group of an adenosine (A)
residue (known as the branch site) in the
intron attacks and forms a phosphodiester
bond with the phosphate at the 5' end of the
intron 1
27Biochemistry For Medics- Lecture Notes
28. Splicing of m-RNA
• The newly- feed 3'OH of the
upstream exon 1 then forms a
phosphodiester bond with the
5'end of the downstream exon
2.
• The excised intron is released
as a "lariat" structure, which is
degraded
• After removal of all the
introns, the mature m RNA
molecules leave the nucleus
by passing in to the cytosol
through pores in to the
nuclear membrane.
28Biochemistry For Medics- Lecture Notes
29. Clinical significance of Splicing
1) Antibodies against
snRNPs
In systemic Lupus
Erythematosus (SLE), an
auto immune disease, the
antibodies are produced
against host proteins,
including sn RNPs.
29Biochemistry For Medics- Lecture Notes
30. Clinical significance of Splicing
2) Mutations at the splice site
• Mutations at the splice site can lead to
improper splicing and the production of
aberrant proteins .
• For example some cases of Beta thalassemia
are as a result of incorrect splicing of beta
globin m- RNA due to mutation at the splice
site.
30Biochemistry For Medics- Lecture Notes
31. Biological significance of Splicing
Alternative Splicing
• Alternative patterns of RNA splicing is
adapted for the synthesis of tissue-specific
proteins.
• The pre-m RNA molecules from some genes
can be spliced in two or more alternative
ways in different tissues.
• This produces multiple variations of the m
RNA and thus diverse set of proteins can be
synthesized from a given set of genes
31Biochemistry For Medics- Lecture Notes
32. Biological significance of Splicing
Alternative Splicing
• For example- Tissue specific tropomyosins
are produced from the same primary
transcript by alternative splicing.
• Alternative splicing and
processing results in the formation of seven
unique α -tropomyosin mRNAs in seven
different tissues
32Biochemistry For Medics- Lecture Notes
33. Biological significance of Splicing
• Tissue specific proteins are produced from the same
primary transcript by alternative splicing
33Biochemistry For Medics- Lecture Notes
34. Biological significance of Splicing
• Similarly, the use of alternative termination-
cleavage-polyadenylation sites also results in
mRNA variability .
• Alternative polyadenylation sites in the
immunoglobulin heavy chain primary transcript
result in mRNAs that are either 2700 bases long
(m) or 2400 bases long (s).
• This results in a different carboxyl terminal region
of the encoded proteins such that the m protein
remains attached to the membrane of the B
lymphocyte and the s immunoglobulin is secreted.
34Biochemistry For Medics- Lecture Notes
35. Biological significance of Splicing
• By means of alternative poly A sites variability in
mRNA can be produced and thus different proteins
can be synthesized from a given set of genes
35Biochemistry For Medics- Lecture Notes
36. Inhibitors of Transcription
• Rifampicin- binds with Beta subunit of
prokaryotic RNA polymerase,
• It is an inhibitor of prokaryotic
transcription initiation.
• It binds only to bacterial RNA
polymerase but not to eukaryotic RNA
polymerases.
• Therefore, Rifampicin is a powerful drug
for treatment of bacterial infections.
• Used for the treatment of tuberculosis
and leprosy
36Biochemistry For Medics- Lecture Notes
37. Mechanism of action of
Actinomycin D
• Actinomycin D- Intercalates with DNA
strands
• Actinomycins inhibit both DNA
synthesis and RNA synthesis by
blocking chain elongation.
• They interact with G·C base pairs as
they require the 2-amino group of
guanine for binding.
• Actinomycins are used as anticancer
drugs
Biochemistry For Medics- Lecture Notes 37
39. Alpha amanitin
• Alpha amanitin is a molecule made
from the “death cap” mushroom
and is a known potent inhibitor
RNA polymerase.
• One single mushroom could very
easily lead to a fast death in 10
days.
• The mechanism of action is that
alpha amanitin inhibits RNA
polymerase –II at both the
initiation and elongation states of
transcription.
Biochemistry For Medics- Lecture Notes 39
40. Revision of concepts
• Follow the link
http://highered.mcgraw-
hill.com/olcweb/cgi/pluginpop.cgi?it=swf::
535::535::/sites/dl/free/0072437316/120
077/bio30.swf::How%20Spliceosomes%20
Process%20RNA
40Biochemistry For Medics- Lecture Notes