Transcript Part 1

Structure & functions of RNA
Ribonucleic acid (RNA) is a long polymer of nucleic
acid monomers that is structurally similar to DNA but
has a vast array of diverse functions, the most
important being its central role in protein synthesis.
Deepa John
Harini Chandra
Affiliations
Master Layout (Part 1)
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This animation consists of 3 parts:
Part 1 – Structure of RNA
Part 2 – Different classes of RNA
Part 3 – Functions of different classes RNA in protein synthesis
Sugar
phosphate
backbone
Nucleoside
Nitrogenous
bases –
Adenine/
Guanine/
Uracil/
Cytosine
Base
Ribose sugar
Phosphate P
Base
Ribose sugar
5
Nucleotide
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Definitions of the components:
(Part 1 – Structure of RNA)
1. Nucleoside: A base bound to a sugar, either ribose or deoxyribose, by
means of a b-glycosidic linkage.
2. Nucleotide: The subunit or chain link in DNA or RNA composed of a
sugar, a base and at least one phosphate group. It is more specifically
known as ribonucleotide in RNA.
3. Ribonucleic acid (RNA): A polymer composed of ribonucleotides, linked
together by phosphodiester bonds.
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4. Ribose sugar: A monosaccharide, aldopentose sugar that is abundant in
nature in its D isomeric form and is the sugar component of RNA.
5. Pyrimidine: An organic compound similar to benzene and pyridine that
is composed of a heterocyclic, aromatic six-member ring having nitrogen
atoms at positions 1 and 3. The nitrogenous bases found in RNA, cytosine
and uracil, are derivatives of pyrimidine.
6. Purine: These are the most abundant nitrogen-containing heterocyclic
compounds in nature and are composed of a pyrimidine ring fused with an
imidazole ring. Derivatives of this aromatic compound occur in both DNA &
RNA in the form of adenine and guanine.
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Definitions of the components:
(Part 1 – Structure of RNA)
7. Adenine (A): This is a purine nucleobase found in both DNA and RNA
that pairs with thymine (T) through two hydrogen bonds in the double
stranded RNA (dsRNA) structure. In addition to being a component of
genetic material, it is also essential for synthesis of various cofactors in the
body.
8. Guanine (G): This is another purine nucleobase found in both DNA and
RNA. This planar, bicyclic molecule base pairs with cytosine in dsRNA.
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9. Cytosine (C): This pyrimidine derivative found to be a component of
both DNA and RNA base pairs with guanine in the dsRNA structure.
10. Uracil: This pyrimidine derivative found to be a component of only RNA
base pairs with adenine in the dsRNA structure.
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1 Part 1,Step 1:
Nucleoside
Base
Base
Base
Base
2
Polynucleotide chain
Base
Phosphodiester
bond
3’
5’
3’
P
P
5’
3’
5’
P
P
3
P
5’
5’
3’
3’
Nucleotide
Ribose sugar
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Action
As shown
in
animation
.
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Description of the action
First show the pink pentagon below with label,
followed by blue ‘base’ being attached .The curly
bracket must appear with the label ‘nucleoside’.
Then the ‘pink circle as depicted in the animation
must appear followed by the next curly bracket
and label ‘nucleotide’. The thick black line must
then appear with label followed immediately by
the next unit and so on.
Audio Narration
RNA is made up of three basic components – a sugar, a
nitrogenous base and a phosphate group. The sugar and base
are linked to form a nucleoside and attachment of the
phosphate group results in a nucleotide. Many such nucleotide
units are linked together by means of a covalent bond known
as the phosphodiester bond. This is formed between the 3’
carbon of one sugar and 5' carbon of the next sugar via a
phosphate group to give rise to a polynucleotide chain.
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Part 1, Step 2:
Purines
Pyrimidines
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Action
5
Show the
structures
above with
their labels
and numbering
as depicted.
Description of the action
(Please use black background &
redraw all figures)
Make the four structures appear
one at a time as depicted with
their labels and numbering
around the structure.
Audio Narration
RNA is composed of four different nitrogenous
bases that are derivatives of the heterocyclic,
aromatic compounds, purines and pyrimidines.
Adenine and guanine are purines while uracil and
cytosine are the pyrimidines.
Source: www.mun.ca/biochem/courses/3107/.../bases_and_chains.html, www.bio.miami.edu
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Part 1, Step 3:
5’ - U U G G U G G A G U C U G C A A C U G A C U C C A U U G C A - 3'
Single stranded RNA
Single stranded RNA tends
to assume right handed
helical confirmation
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3
Bases are shown in gray.
Phosphate atoms in yellow and
ribose in green.
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RNA secondary structure
Action Description of the action
5
A single stranded RNA
molecule may fold back on
itself to form a secondary
structure (stem and loop)
Show all
the
forms of
RNA
(Please use black background & redraw all figures)
First show the linear sequence of alphabets on top.
Next show the left arrow, the respective text followed
by the winding of the sequence of top around a
vertical axis to give the figure on the left bottom. Then
show the right arrow and respective text followed by
the bending of the sequence to give the right bottom
figure with the alphabets in red next to each other as
shown.
Audio Narration
RNA exists mainly as a single-stranded molecule. The base stacking
interactions often tend to make the RNA assume a right-handed
helical conformation. Single stranded RNA also forms secondary
structures by folding back on itself resulting in formation of loops and
hairpins due to base pairing interactions. Functional RNA molecules
often require a specific tertiary structure, the scaffold of which is
provided by the secondary structure. These RNA due to their large
negative charge are stabilized by metal ions.
Source: Biochemistry by Lehninger, 4th edition (ebook), Biochemistry by Stryer,5th edition(ebook)
Master Layout (Part 2)
1
This animation consists of 3 parts:
Part 1 – Structure of RNA
Part 2 – Different classes of RNA
Part 3 – Functions of different classes RNA in protein synthesis
Acceptor stem
Large subunit
2
TyC loop
D loop
Small subunit
Ribosomal RNA (rRNA)
Variable loop
3
Anticodon loop
5’
3’
Transfer RNA (tRNA)
Messenger RNA (mRNA)
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Cap 5‘UTR Start Coding sequence
5
Stop 3‘UTR
Poly A tail
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3
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Definitions of the components:
Part 2 – Different classes of RNA
1. mRNA: The messenger RNA is a long sequence of nucleotides that serves
as a template for protein synthesis. It is transcribed from a DNA template by
RNA Polymerase and gets translated into the amino acid sequence of the
corresponding protein. Eukaryotic mRNA requires extensive processing to
form the mature mRNA while prokaryotic mRNA does not. Typical mRNA
structure is composed of the following regions:
a) Cap: An altered nucleotide consisting of a methylated guanosine residue
bound through 5’ - 5’ triphosphate linkage to the first transcribed nucleotide of
the mRNA. Capping takes place only in eukaryotes and is a vital process to
produce mature mRNA and provide a recognition site for binding of ribosomes.
b) 5’ UTR: An untranslated region in mRNA that is present before the start
codon and plays an important role in providing mRNA stability, facilitating
mRNA localization and providing a ribosome binding site.
c) Start site: It is the site from where translation is initiated. The codon at this
site is usually AUG coding for methionine.
d) Coding sequence: These are the regions in mRNA that encode specific
amino acid sequences for the process of translation into a polypeptide chain. It
begins with a start codon and ends with a stop codon.
e) Stop site: The site where translation is terminated. The codon here is
usually UAA, UAG and UGA. These do not code for any amino acids.
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Definitions of the components:
Part 2 – Different classes of RNA
f) 3’ UTR: An untranslated region in mRNA that is present after the stop codon.
It has several roles including mRNA stability and localization.
g) Poly A(poly adenylic acid) tail: A sequence of about two hundred adenine
residues added to the end of eukaryotic mRNA that plays an important role in
nuclear export, translation and providing stability to the mRNA.
2. tRNA- A relatively small RNA molecule involved in protein synthesis that
binds an amino acid at one end and base pairs with an mRNA codon at the
other, thus serving as an adaptor that translates an mRNA code into a
sequence of amino acids. A tRNA molecule consists of the following
components:
a) Acceptor stem: This is a stem of around 7 base pairs that is formed by base
pairing of the two ends of tRNA, the 5'-terminal nucleotide with the 3'-terminal
nucleotide.
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b) TyC loop:This is a 5 base pair stem containing the sequence TΨC in which
the Ψ stands for a modified nucleotide, pseudouridine. Pseudouridine is similar
to normal uridine except that the base is linked to the ribose through 5th carbon
of the base instead of the nitrogen-1.
c) Variable loop: The region between the anticodon loop and T loop which is
so called because it varies in length from 13 to 14 nucleotides.
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Definitions of the components:
Part 2 – Different classes of RNA
d) Anticodon loop: The loop, conventionally drawn at the bottom of tRNA
molecule, that contains a 3 base sequence that base pairs with a specific
codon of mRNA.
e) D-loop: It is dihydrouracil loop made up of a 4 base pair stem. It is so
named because of the modified uracil base that the region contains.
3. rRNA- rRNA forms the central component of ribosomes. It has both
catalytic and structural roles in protein synthesis. The ribosome that houses
this rRNA consists of a large subunit and a small subunit.
Part
2,
Step
1
1
Mature mRNA structure
5’ Cap
5‘UTR
Start
Coding sequence
Stop
3‘UTR
Poly A tail
3’
5’
2
AUG
UAG/UAA/UGA
AAAAAAAAAAA
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Action Description of the action
5
As
shown
in
animati
on.
(Please use black background & redraw all
figures)
Show the pale green ‘mRNA’ structure which
is present in the background. Next, each
region must be highlighted sequentially with
the label for that particular region appearing.
In places indicated above, the details of the
structure of the highlighted region must be
shown in a zoom box as depicted in the
animation.
Audio Narration
Messenger RNA is formed from a DNA template by transcription. This mRNA
is often referred to as the pre-mRNA in eukaryotes since it undergoes further
processing to form a mature mRNA. A fully processed eukaryotic mRNA
includes a 5’ cap, where the nucleotide at the 5’ end is modified by addition of
7-methyl guanosine and a poly A tail at the 3’ end which serves to protect the
mRNA from degradation by exonucleases. The mRNA also contains 5’ and 3’
UTRs that contain signal sequences and serve as binding sites for various
proteins. The coding sequence is flanked by start and stop codons that define
the beginning and end of the gene to be transcribed.
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Part 2, Step 2
tRNA processing & structure
Dihydrouridine
Modified
nucleotide
bases in
tRNA
Acceptor stem
RNAses
2
TyC loop
v
D loop
Variable
loop
3
Anticodon loop
Pseudouridine
Primary transcript
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Action
First show
image on left
and yellow
segments being
removed
followed by
folding to form
image in the
centre & then
right.
Mature tRNA
Secondary structure of tRNA
Description of the action
(Please use black background & redraw all figures) First
show the figure on the left followed by the colored
‘RNAses’. These must cut out the yellow regions of the
figure which must then disappear. The green portion at
the bottom must join with the hanging end on the other
side to form a loop as shown in the centre panel and a
pink region on the right top of the figure must be added.
Two of the zoomed in structures must then be shown
and finally, this structure must be shown to fold on itself
and the figure on right must appear.
Tertiary structure of tRNA
Audio Narration
Longer RNA precursors are modified by enzymatic removal of
nucleotides from the 5’ and 3’ ends to form the tRNA structure.
Additional processing of the tRNA such as attachment of the 3’
CCA trinucleotide unit and modification of certain bases takes
place in ceratin bacteria and almost all eukaryotes. All tRNAs have
a common secondary structure represented by a clover leaf having
four base-paired stems. The anticodon loop recognizes the
corresponding mRNA codon while the acceptor stem adds the
suitable amino acid to the growing polypeptide chain.
Source: Biochemistry by Lehninger, 3rd edition (ebook); www.sparknotes.com,molecular biology by Rober Weaver.
Part
2,
Step
3
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Prokaryotic ribosome
Large subunit - 50S
Methylation
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3
Pre-rRNA transcript (30S)
16S
23S
tRNA
23S
5S
Cleavage by specific
RNAses & nucleases
Methylated sites
on rRNA
Small subunit - 30S
16S
Mature rRNAs
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Action Description of the action
5
tRNA
As
shown
in
animati
on.
(Please use black background & redraw all figures) First show the
two units on the left and then the larger unit moving down and joining
the smaller one. Next the small unit must be zoomed into and the
figure on top right must appear with labels. The small orange circle
must then appear and traverse the length of the rectangle up to the
end of ‘23S’. As it moves, small green stars must appear as shown in
the middle figure. Next the pie-shaped objects must appear at the
sites indicated and must cut through the rectangle. These blue
portions must then be removed and the bottom figure must appear.
Source: www.ncbi.nlm.nih.gov
5S
Audio Narration
rRNA is the central component of the ribosome
involved in protein synthesis in all living cells.
Prokaryotic 70S ribosome is composed of 50S and 30S
subunits where S is a measure of the rate of
sedimentation of the respective components in a
centrifuge. rRNAs are derived from longer precursors
called pre-rRNA. A single 30S rRNA precursor is
processed by several enzymes to give rise to 16S, 23S
and 5S rRNAs in bacteria.
Part
2,
Step
4
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Eukaryotic ribosome
Pre-rRNA transcript (45S)
18S
Large subunit - 60S
5.8S
28S
Cleavage by small
nucleolar RNAs
Methylated sites
on rRNA
Small subunit - 40S
18S
Mature rRNAs
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Action
5
28S
Methylation
2
3
5.8S
As
shown
in
animati
on.
Description of the action
(Please use black background & redraw all figures) First show the
two units on the left and then the larger unit moving down and joining
the smaller one. Next the small unit must be zoomed into and the
figure on top right must appear with labels. The small orange circle
must then appear and traverse the length of the rectangle up to the
end of ‘23S’. As it moves, small green stars must appear as shown in
the middle figure. Next the pie-shaped objects must appear at the
sites indicated and must cut through the rectangle. These blue
portions must then be removed and the bottom figure must appear.
Source: www.ncbi.nlm.nih.gov
Audio Narration
Eukaryotic 80S ribosome is composed of 60S and 40S
subunits where S is a measure of the rate of
sedimentation of the respective components in a
centrifuge. In eukaryotic vertebrates, a single 45S
rRNA precursor is processed by several enzymes to
give rise to 18S, 5.8S and 28S rRNAs.
Master Layout (Part 3)
1
This animation consists of 3 parts:
Part 1 – Structure of RNA
Part 2 – Different classes of RNA
Part 3 – Functions of different classes of RNA in protein synthesis
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3
Growing
polypeptide chain
Amino acid
Ribosome
Outgoing tRNAs
Incoming
aminoacyl-tRNAs
Start codon
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5
5’
A site
P site
mRNA
Movement of
ribosome
3’
Stop codon
1
Definitions of the components:
Part 3 – Functions of different classes of RNA in
protein synthesis
2
1. 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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2. mRNA: The messenger RNA is the intermediate between DNA and the
protein which encodes the required “blueprint” of the protein product. The RNA
obtained from DNA immediately after transcription is known as the pre-mRNA
and is made up of both coding (exons) and non-coding (introns) regions. This
mRNA is further processed to give the mature mRNA which contains coding
and other essential sequences for protein synthesis.
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3. Ribosome: An RNA- protein particle that is involved in translation of the
mRNA into protein. Prokaryotic 70S ribosome is composed of 30S and 50S
subunits, while eukaryotic 80S ribosome is composed of 40S and 60S
subunits, where S is a measure of the rate of sedimentation of the respective
components in a centrifuge (Svedberg).
4. Incoming aminoacyl tRNAs: The tRNA carrying the amino acid specified
by the next subsequent codon on the mRNA chain.
5. Outgoing tRNAs: The uncharged tRNA exiting from the ribosome after
transferring the amino acid to the growing polypeptide chain attached to the
tRNA in the P site.
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2
Definitions of the components:
Part 3 – Functions of different classes of RNA in
protein synthesis
6. Growing polypeptide chain: A single protein chain that progressively
increases in length during translation until it reaches the stop codon. It is
composed of specific sequence of amino acids linked together by peptide
bonds.
7. Start codon: The position at which initiation of translation takes place and
most often contains the codon AUG that codes for methionine.
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8. Stop codon: The site that codes for the termination of translation and
consists of one of the three codons - UAA, UAG or UGA.
9. A site: The site on the ribosome to which the charged, incoming amino
acyl-tRNA (except the first one) binds.
10. P site: The site on ribosome to which the peptidyl-tRNA is bound at the
time that a new amino acyl-tRNA enters the ribosomal A site.
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Part
3,
Step
1
1
Initiation
Large ribosomal
subunit
2
3’
Stop codon
Start codon
5’
3
mRNA
Initiator fMet
tRNA
P site A site
Small ribosomal subunit
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Action Description of the action
As shown
in
animation.
5
(Please use black background )First
show the small unit at the bottom
followed by binding of the blue strand to
this pink unit. The marked AUG
sequence must fall in the ‘P site’. Next
the blue figure on top which must move
down and bind to the ‘AUG’ below. Then
the large pink subunit must be
assembled on top of these.
Audio Narration
Initiation of protein synthesis is carried out by binding of the mRNA
to the small ribosomal subunit such that its initiation codon, most
often an AUG sequence, is aligned at the P site. The initiator tRNA
that carried a modified methionine amino acid on its acceptor stem
then binds to the ribosomal subunit by means of codon-anticodon
interactions. The large subunit is then assembled on top of this to
form the initiation complex. Other initiation factors are also
involved which ensure correct positioning of all the components.
Part
3,
Step
2
1
Elongation
Incoming aminoacyl tRNAs
2
Peptide bond formation
Large ribosomal subunit
Initiator fMet
tRNA
3
3’
Stop codon
Start codon
5’ P site A site
mRNA
Small ribosomal subunit
4
Action Description of the action
As shown
in
animation.
5
(Please use black background )
The colored figures on top must be
shown to enter the frame and then the
orange figure must move down and bind
to the ‘A site’. Once this happens, the
blue circle in the ‘P site’ must move and
bind on top of the orange circle in the ‘A
site’. Next the blue figure must be moved
out of the P site as depicted in animation.
Audio Narration
The next incoming aminoacyl tRNA carrying the amino acid
corresponding to the next codon occupies the A site. A peptide
bond is then formed between the amino acid in the A site and the P
site with the P site amino acid beingtransferred to the A site. The
unbound tRNA then leaves the P site and is moved to the exit or E
site briefly before being removed.
Part
3,
Step
3
1
Elongation
Incoming aminoacyl tRNAs
2
Peptide bond formation
Large ribosomal subunit
Initiator fMet
tRNA
3
4
5
Start codon P site A site
5’
Small ribosomal subunit
Action
Description of the action
(Please use black background )
As shown
The pink subunits must move a little bit to the right such
in
that the orange figure with blue circle on top now
animation.
occupies the ‘P site’. Then the next green figure on top
must enter the ‘A site’ and the same sequence of events
mentioned in step 2 must be repeated such that the
orange and blue circles are transferred on top of the
green circle and the empty orange figure leaves the
figure. Then again the pink subunits must move and so
on until many colored circles have been added.
3’
Stop codon
mRNA
Audio Narration
Once the peptide bond has been formed, the
ribosome moves one codon towards the 3’ end of
the mRNA such that the tRNA in the A site now
occupies the P site and the A site is again free for
the next incoming aminoacyl tRNA. Multiple such
rounds of elongation followed by translocation of
the tRNAs are carried out to form the growing
polypeptide chain.
Part
3,
Step
4
1
Termination
Polypeptide chain
released
Dissociation of all
components
2
Large ribosomal subunit
Release factor
3
3’
Stop codon
Start codon
5’
mRNA
P site A site
Small ribosomal subunit
4
5
Action
Description of the action
As shown (Please use black background )
Once several circles have been added and the pink
in
animation. subunits reach the ‘stop’ signal on the blue chain, an
oval shape must enter the ‘A site’ with the label
‘release factor’. Once this happens, the chain of
circles must be dissociated from the last colored
‘tRNA’ figure (red rectangular figure above). After this,
all the other components i.e. the pink units and the
blue chain must also move apart to show that they
are dissociating as depicted in the animation.
Audio Narration
When the ribosome encounters the termination
sequence, typically UAA, UAG, UGA, a release
factor binds to the vacant A site and the
polypeptide chain is hydrolyzed and released.
Other termination factors also aid this process.
Once synthesis is complete, the ribosomal
subunits dissociate from each other and all
components are separated until commencement
of the next round of translation.
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Interactivity option 1:Step No:1
What inference can be drawn from this experiment?
2
A) Subunit exchange between ribosomes does not occur after each round of translation.
B) Subunit exchange between ribosomes occurs after each round of translation.
C) One of the two subunits had degraded
3
D) Only one of the two subunits dissociates.
4
Interacativity Type
Choose the
correct option
5
Options
(Please use black
background ) User
must be allowed to
choose one of the four
options after the
experiment displayed
in the next slide is
shown.
Boundary/limits
Results
B is the correct answer,. If user
chooses B, it must turn green
with the remark ‘correct
answer’. If user chooses any of
the remaining options, it must
turn red with the remark
‘incorrect answer’.
Interactivity option 1:Step No:2
1
The following experiment was performed….
After 3.5 generations, isolate
the riobosmes and analyze by
sucrose density gradient
centrifugation with 14C labeled
riobosomes for comparison.
2
3
Label ribosome by growing
E. coli in presence of heavy
isotopes like N, C and H and
make them radioactive by
including some 3H.
Place cells with labeled
heavy ribosomes in
medium with ordinary
light isotopes of N, C and
H.
Labeled whole
ribosome had a
hybrid sedimentation
coefficient in between
the light and heavy
ribosome
sedimentation
coefficient
4
+
5
Heavy ribosomes
radioactively
labeled(indicated
by asterisk sign)
Heavy
ribosome
subunit
Light
ribosome
Labeled
Whole
Ribosome
(hybrid
sedimentation
coefficient)
1
Questionnaire
1. The form of genetic information used directly in protein synthesis is
Answers: a) DNA b) mRNA c) rRNA d) ribosomes
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2. The process in which ribosome get engaged
Answers: a) transcription b) translation c) replication d) cell division
3. The bases of RNA are the same as those of DNA with the exception that RNA contains
3
4
Answers: a) cysteine instead of cytosine b) uracil instead of thyminec) cytosine instead of
guanine d) uracil instead of adenine
4. The nucleotide sequences on DNA that actually have information encoding a sequence
of amino acids are
Answers: a) introns
c) UAA
d) UGA
5. Which one of the following is not a type of RNA?
Answers: a) rRNA
5
b) exons
b)nRNA
c)mRNA
d) tRNA
Links for further reading
Books:
Genetics by Peter.J.Russell, 5th edition
Molecular biology by Robert Weaver, 4th edition
Biochemistry by Lubert Stryer et al., 6th edition (ebook)
Molecular Biology of the Gene by James Watson et al., 5th edition