Transcript Part 1

Genomics to proteomics
The genome represents only the starting point
towards understanding complexity of biological
functions. The products of gene expression, proteins,
provide a much more meaningful insight into the
mysteries of essential biological processes.
Harini Chandra
Affiliations
Master Layout
1
2
2. Single gene, multiple
proteins
3
1. Genomic DNA contains
large stretches of non-coding
regions
4
Action
5
Show the
figure above
along with the
headings
which the
user must be
able to click
on.
Description of the action
The figure above must be shown with each of
the headings appearing. The headings in red
are the three tabs for each animation described
in the subsequent slides. The user must be
allowed to click on these tabs which must lead
to the corresponding animation for that heading.
When the user moves the cursor over any of the
remaining marked regions, the definition for that
part must popup as given in the next slide.
3. Post-translational
modification of proteins
Audio Narration
The genome represents only the starting point towards understanding
complexity of biological functions. The products of gene expression,
proteins, provide a much more meaningful insight into the mysteries of
essential biological processes. The number of proteins expressed in a
cell is much larger than its genomic counterpart due to the posttranscriptional and post-translational modifications taking place. To
obtain a clear understanding of cellular processes & regulation, there
has been a gradual shift in focus from the genome to the proteome due
to the large amount of information available from the proteome.
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2
Definitions of the components:
Master layout
1. Genome: The entire sequence of an organism’s hereditary information,
including both coding and non-coding regions, encoded in DNA is known as
the genome.
2. Transcriptome: The set of all RNA molecules, including mRNA, rRNA
and tRNA, present in an organism is referred to as the transcriptome.
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4
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3. Proteome: The entire complement of proteins expressed by the genome
of an organism under specific defined conditions is known as the proteome.
Like the transcriptome, the proteome of an organism will also vary with
external factors and conditions.
Master Layout (Part 1)
1
2
This animation consists of 3
parts:
Part 1 – Genomic DNA
contains large stretches of
non-coding regions
Part 2 – Single gene, multiple
proteins
Part 3 – Post-translational
modification of proteins
5’
Genomic DNA
Transcription
Exon 1
Intron
Pre-mRNA
3’
Exon 2
3
5’
4
3’
Spliceosome
assembly
Lariat
Exon 1
5
Exon 2
Mature mRNA
Biochemistry by Stryer et al., 5th edition (ebook)
1
2
3
4
5
Definitions of the components:
Part 1 – Genomic DNA contains large stretches of
non-coding regions
1. Genomic DNA: The deoxyribonucleic acid polymeric sequence that acts
as the store for genetic information and is essential for the synthesis of RNA
and protein molecules, which are necessary for cellular functioning in all
organisms.
2. Transcription: The process by which the genomic DNA is converted into
a chemically related molecule, the messenger RNA or mRNA. Several
enzymes and other factors are involved in this process. All regions of the
DNA, coding and non-coding, get transcribed into the corresponding mRNA.
3. Pre-mRNA: The mRNA transcript that is produced from DNA as soon as
transcription ends is known as the pre-mRNA. This contains both coding
and non-coding sequences, is short lived and is further processed before
translation.
4. Exon: The regions of the mRNA that code for specific portions or entire
protein products upon translation are known as exons. They are often
discontinuous with intervening nucleic acid sequences being present
between them.
5. Intron: The intragenic sequences, sometimes considered as “junk”, in the
pre-mRNA that do not get translated into proteins are known as introns.
These are removed during the process of RNA splicing.
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2
3
4
5
Definitions of the components:
Part 1 – Genomic DNA contains large stretches of
non-coding regions
6. Spliceosome assembly: This is made up of several protein-RNA
complexes that bind the pre-mRNA and carry out splicing of the introns to
give the mature mRNA. They are also commonly known as “snRNPs” or
small nuclear ribonucleoproteins. These recognise specific sequences
within the intron where they bind.
7. Lariat: The intron sequence that is removed by the spliceosome
assembly forms a loop like structure with its 5’ end bound to an internal
branch-point adenine residue. This is referred to as the lariat.
8. Mature mRNA: The mRNA that consists of only those nucleic acid
sequences that are translated into proteins is known as the mature mRNA.
1
Part 1, Step 1:
Transcription
RNAP
Genomic DNA
5’
3’
3’
2
5’
Transcription
factors
Template
strand
Gene
5’
3
Pre- mRNA
released
5’
3’
3’
RNAP
3’
5’
mRNA
transcript
RNAP dissociates
5’
3’
RNAP
3’
5’
4
Action
Description of the action
As shown in First show the two blue strands on top followed by the
colored circles appearing on the left of the strands. Next
animation.
5
Audio Narration
Pre-mRNA is synthesized from genomic DNA by the
process of transcription. The gene to be transcribed is
the red oval labelled ‘RNAP’ must bind to the blue strands. bound by transcription initiation factors and then by RNA
The strands must separate as shown in the middle panel Polymerase which transcribes the gene in the 5’ to 3’
and the RNAP oval must move across it. As it moves, the direction. The DNA strand that gets transcribed is
green strand shown must continuously appear behind it.
known as the template strand. Once the termination
This must continue until the RNAP reaches the end. Here
sequences are reached, the enzyme and the newly
it must dissociate from the blue strands along with the
formed mRNA transcript are released.
completed green strand as depicted.
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Part 1, Step 2:
Spliceosome
assembly
Intron
5’
2
Exon 1
GU
A
Exon 2
Pre-mRNA
AG
3’
Recognition elements for
spliceosome assembly
5’
3
Conformation change
3’
4
Action
5
The chain
should change
conformation as
shown after
binding of the
red and purple
shapes.
Description of the action
Audio Narration
(Redraw all figures shown)
First show the coloured intertwined chain on top
followed by the arrow.Then show the tricolour
chain followed by the alphabets and its label.
Next, show binding of the red and purple shapes
at the site shown. Once bound, the shape of the
chain must change as shown below.
The genomic DNA that gets transcribed into mRNA
contains both exons, the coding sequences, as well as
introns which are intervening, non-coding sequences .
This pre-mRNA has certain recognition sites within its
intron sequence that allows the spliceosome assembly
to recognize it and bind to it. There is a conformation
change that takes place upon binding of the proteinRNA complex.
Biochemistry by Stryer et al., 5th edition (ebook)
1
Part 1, Step 3:
Binding of all snRNPs
5’
2
3’
Cleavage at 5’
end of intron
Exon 1 with free 3’ end
5’
3
4
5
Lariat
3’
Action
The pink and
light blue
circles must
then bind
followed by
cleavage at the
site indicated.
Description of the action
The pink and light blue circles must also be shown to
bind to the assembly.
Next, there must be a break occurring at the site
indicated and the green rod must be shown free at that
end.
Audio Narration
The remaining snRNPs bind following
the conformation change of the premRNA and there is cleavage at the GU
site on the 5’ end of the intron. This then
attaches to the branch site adenine
nucleotide near its 3’ end to form the
lariat structure.
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Part 1, Step 4:
Binding of 3’ end of exon 1
to 5’ end of exon 2
2
5’
3’
Cleavage at 3’
end of intron
3
Degraded
5’
4
5
Unlike DNA, Proteins do not contain
intervening sequences and are directly
indicative of cellular function.
Exon 1
Action
The blue circular
structure must get
detached at the
other end also as
shown and the
green and purple
ends must join.
Mature mRNA
Exon 2
Description of the action
First show the blue circle being broken at the other end
also as shown.
The green rod then binds the purple rod as indicated by
the arrow mark.
The blue circle along with the other group of shapes is
removed as shown.
3’
Audio Narration
The assembly then cleaves the 3’ end of
the intron sequence containing the AG
recognition element. The free 3’ hydroxyl
group of the first exon attacks the 5’ end
of the second exon such that they are
joined to give the mature mRNA.
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Master Layout (Part 2)
This animation consists of 3 parts:
Part 1 – Genomic DNA contains large stretches of non-coding regions
Part 2 – Single gene, multiple proteins
Part 3 – Post-translational modification of proteins
Pre-mRNA
2
Exon 1
Intron
Exon 2
Intron
Exon 3
Intron
Exon 4
Alternative
splicing
3
Exon 1
Exon 2
Exon 4
Exon 1
Mature mRNA-A
Exon 3
Mature mRNA-B
Translation
4
Protein A
5
Protein B
Exon 4
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Definitions of the components:
Part 2 – single gene, multiple proteins
2
1. Alternative splicing: This is a process by which the exons or coding
sequences of the pre-mRNA produced by transcription of a gene are
combined in different ways during RNA splicing. The resulting mature
mRNA give rise to different protein products by translation, most of which
are isoforms of one another. In this way a single gene can give rise to
multiple protein products.
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2. Translation: A process by which the mRNA sequence is read in the
form of three letter codes known as codons to incorporate the
corresponding amino acids in the growing polypeptide chain with the
active involvement of rRNA, tRNA and several other enzymes.
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5
3. Proteins A, B: The protein products resulting from translation of the
differently spliced mRNA. Mature mRNA with exons 1, 2 and 4 gives rise
to protein A while mRNA with exons 1,3 and 4 gives protein B.
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Part 2, Step 1:
Pre-mRNA
Exon 1
2
Intron
Exon 2
Exon 3
Action
The multicolored
chain shown
above must be
combined in
different ways to
give rise to the
figures below.
Intron
Exon 4
Alternative
splicing
Exon 4
Exon 1
Mature mRNA-A
3
5
Exon 2
Spliceosome
assembly
Exon 1
4
Intron
Description of the action
First show the multicolored chain shown above with its
various label markings. Next show the colored circular
shapes appearing after the red ‘exon 1’. This assembly
must act like a pair of scissors and first cut the red, blue
and green parts of the chain out, which must rejoin
below to give the ‘mature mRNA-A’. Next, from another
intact chain, the red, violet and green parts of the chain
must be cut out and these must rejoin to form the
‘mature mRNA-B’ shown on the right.
Exon 3
Exon 4
Mature mRNA-B
Audio Narration
Pre-mRNA transcribed from genomic DNA is often made up
of several coding exons interspersed by non-coding introns.
Alternative splicing, a common phenomenon observed in
eukaryotes, allows the exons to be reconnected in multiple
ways. There are several mechanisms for alternative splicing,
the most common being exon skipping, wherein a particular
exon may be included in the mature mRNA under specific
conditions or in certain tissues and omitted from others.
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Part 2, Step 2:
Translation
Amino
acid
Ribosome
tRNA
2
Mature mRNA
3’
3’
5’
Movement of
ribosome
5’
Growing
polypeptide chain
3
3’
3’
5’
5’
4
5
Action
The pink
‘ribosome’ must
move along the
blue strand with
the
rectangle+circle
coming in one at
a time.
Description of the action
First show the figure on top left. Next show the blue and orange
rect+circle coming in at position shown in fig 2 along with the other 3
rect+circles coming in slowly from the side. Next, the blue circle must
be transferred on top of orange and blue rect must leave as shown in
fig.3. The pink units must then move towards the right such the orange
rectangle is now in same place where the blue was earlier. Green
rectangle+circle must enter in position of orange and remaining 3 must
slowly come in. This displacement must continue to happen as shown
in fig.4 with the circles being continuously added on top of each other.
Audio Narration
The mature mRNA produced then undergoes
translation where it is bound to the ribosome
and read as three letter codons. The
corresponding amino acids are incorporated
with the help of tRNAs. The ribosome moves
along the mRNA and continues to incorporate
the amino acids to the growing polypeptide
until the termination codon is reached.
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Part 2, Step 3:
Exon 1
Exon 2
Exon 4
Translation
Mature mRNA-A
Protein A
2
Exon 1
3
4
5
Exon 3
Exon 4
Translation
Mature mRNA-B
Protein B
The proteome is significantly more complex than
the genome since a single gene can encode
multiple proteins. Without studying the proteome,
it will not be possible to understand the conditions
under which a particular protein is expressed.
Action
The colored
chains must
be shown to
give to
different
‘proteins’.
Description of the action
First show the first colored chain giving rise to the blue
‘protein A’ followed by the second colored chain giving
‘protein B’. This must be followed by appearance of the
textbox below.
Audio Narration
The diversity of proteins encoded by a genome
is greatly increased due to alternative splicing.
Each mature mRNA formed gives rise to
different protein products upon translation.
Complexity of the proteome is therefore
understood from the fact the a single gene can
code for multiple proteins.
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Master Layout (Part 3)
This animation consists of 3 parts:
Part 1 – Genomic DNA contains large stretches of non-coding regions
Part 2 – Single gene, multiple proteins
Part 3 – Post-translational modification of proteins
2
Posttranslational
modification
Protein
Hydroxylation
Phosphorylation
Glycosyl
ation
3
P
Methylation
OH
P
OH
CH3
P
4
P
Glucose
Glucose
CH3
Glucose
P
Glucose
Modified proteins
5
CH3
CH3
CH3
OH
OH
1
2
3
4
Definitions of the components:
Part 3 – Post-translational modification of proteins
1. Protein: The polypeptide chain made up of several amino acid residues gets
released at the end of the translation process and undergoes appropriate folding into
its secondary and tertiary structures. If the protein is made up of multiple subunits,
these come together to form the native protein structure.
2. Post-translational modification (PTM): Many proteins undergo chemical
modifications at some of their amino acid residues after translation. These are carried
out by enzyme catalyzed reactions and are essential for normal functioning of the
protein. Some of the most commonly observed PTMs include:
a) Phosphorylation: The addition of a phosphate group, usually to serine, threonine,
tyrosine or histidine residues of the protein. Protein phosphorylation and
dephosphorylation is one of the most important control mechanisms for interconversion of proteins between their functional and non-functional states.
b) Glycosylation: The enzymatic addition of saccharides to specific amino acid
residues resulting in the formation of glycoproteins. Sugars like glucose and mannose
are commonly added to either nitrogen atoms of aspargine, arginine or to hydroxyl
oxygen atoms of serine, threonine, tyrosine etc.
c) Methylation: Addition of a methyl group, usually at lysine or arginine residues.
d) Hydroxylation: Addition of a hydroxyl (-OH) group by the hydroxylase enzymes.
Proline is usually the principal residue that is hydroxylated resulting in hydroxyproline,
an essential and abundant component of connective tissues like collagen.
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3. Modified protein: The protein that has undergone the required PTMs and is ready
to function is the modified protein in its native, stable state conformation.
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Part 3, Step 1:
The final functional protein structure most
often does not correlate directly with the
genome sequence due to several PTMs
made in the protein. This further increases
complexity of the proteome.
Posttranslational
modifications
2
Protein
Hydroxylation
Phosphorylation
Glycosyl
ation
P
3
Methylation
OH
P
OH
CH3
P
P
Glucose
Glucose
CH3
CH3
Glucose
P
CH3
OH
OH
CH3
Glucose
4
5
Modified proteins
Action
The blue chain
on top must be
shown to
which the
groups shown
in figures
below must be
added.
Description of the action
Please redraw figure.
First show the blue chain on top. Then show
the various groups shown in the figures below
being added to the blue chain and appearance
of the images below as shown followed by the
text box above.
Audio Narration
The protein obtained by translation undergoes
folding and various PTMs such as
phosphorylation, alkylation, glycosylation,
hydroxylation etc. to give the final functional
protein. This adds to the complexity of each
protein since the functional protein product does
not directly correspond to its gene sequence.
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Interactivity option 1:Step No:1
Vs
2
4. Gives the true
picture of
functioning of an
organism.
GENOMICS
PROTEOMICS
5. Undergoes no further
modifications once in its
functional state.
2. Can give rise to
multiple products which
need to be further
determined through
experiments.
3
6. May not correspond
to the final product
which could be
modified.
3. Contains
large
intervening
sequences.
4
Interacativity Type
Drag and drop
5
1. Expression
levels are directly
related to cellular
activity.
Options
User must be
allowed to drag the
boxes shown above
and drop them
under the correct
column.
Boundary/limits
Results
Once the user drags and drops a
box under one column, it must
turn green if correct or red if
wrong. Box 1, 4 and 5 must come
under ‘Proteomics’ column while
Box 2, 3 and 6 must come under
‘Genomics’ column.
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2
Questionnaire
1. A pre-mRNA sample has 150 nucleotides with two introns and two exons. Both the
introns are 30 nucleotides long. What will be the length of the final mature mRNA?
Answers: a) 100 b) 85 c) 90 d) 150
2. Which enzyme is responsible for the process of transcription?
3
Answers: a) DNA Polymerase b) RNA Polymerase
tRNA synthetase
c) Peptidyl transferase d) Aminoacyl
3. The process of translation does not require which of the following?
Answers: a) mRNA b) Ribosome c) tRNA d) DNA
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5
4. The lariat structure is formed during which of the following processes?
Answers: a) Replication b) Translation c) Transcription d) Splicing
Links for further reading
Books:
Discovering Genomics, Proteomics & Bioinformatics (2nd edition), A.
Malcolm Campbell & Laurie J. Heyer
Research papers:



Pandey, A. & Mann, M. Proteomics to study genes and genomics.
Nature 2000, 405, 837-846.
Phizicky, E., Bastiaens, P. I. H., Zhu, H., Snyder, M., Fields, S.,
Protein analysis on a proteomic scale. Nature 2003, 422,208–215.
Wilkins, M. R., Williams, K. L., Apple, R. D. & Hochstrasser, D. F.
Proteome Research: New Frontiers in Functional Genomics 1–243
(Springer, Berlin, 1997).