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RIBOZYMES
from Chapter 3 and more
Thanksgiving diversion #2
• Did you know they’re
sequencing the turkey
genome?
• They’ve finished the 1x
shotgun sequence phase.
This means they have
sequences for thousands
of fragments scattered
throughout its genome, but
there are many gaps
missing fragments.
most important concept:
Enzymes control chemical reactions.
They replace activation energy and
may help transfer energy to/from ATP.
most important concept:
Enzyme action is determined by enzyme shape;
enzyme shape is controlled by its primary
structure (its amino acid sequence), all part of the
central dogma of modern biology:
DNA--->RNA--->protein --> us
(Francis Crick)
Figure 3.13
A
B
C
Potential energy
Transition state
Ea without
enzyme
Ea with
enzyme
A + BC
Reactants
AB + C
Products
Time
Box 3.3 Figure 1a
Specificity between substrate and enzyme
Substrate
(glucose)
Enzyme
(hexokinase)
ACTIVE SITE
• each enzyme has a four-dimensional space
which is like a “glove” that the substrate fits
into.
most important concept:
Enzyme action is determined by enzyme shape; enzyme shape is controlled by
its primary structure (its amino acid sequence), all part of the central dogma of
modern biology:
DNA--->RNA--->protein --> us
(Francis Crick)
BUT sometimes the enzymes are RNA;
then ribozymes control chemical
reactions. They replace activation energy
and may help transfer energy to/from ATP
Freeman’s theme
Throughout the textbook, the author shows
how the facts of biology and chemistry
support evolution, including biochemical
evolution in the early chapters--
The RNA world
Figure 3.15b
Secondary structure
of RNA
Stem
Hairpin
Loop
3´
G
G
C
C
U
A
A
U
C
G
5´
Many ribozymes
have been identified and studied
• intron processing
• ribosome activities (protein
synthesis)
• synthetic RNA
• example in textbook chapter 3
Molecules of the “Prebiotic Soup”
• Part of Freeman’s theme
• Nitrogenous bases:
– purines (A, G);
– pyrimidines [C, T, U].
– Purines have been synthesized
in experiments simulating
early earth conditions
(like the Miller/Urey flasks),
but not pyrimidines so far.
Molecules of the “Prebiotic Soup”
Nucleotide monomers
(ATP, GTP, CTP, UTP) =
three phosphate groups
bonded to ribose,
which is bonded to a
nitrogenous base.
• In lab simulations they will form RNA
without enzymes (no polymerases, etc.)
Figure 3.14b
–
O
–
O P O
The sugar-phosphate
spine of RNA
O
5´CH
O
N
2
O
O
O OH
O P O
–
5´
The sequence of
bases found in
an RNA strand
is written in the
5´
3´
direction:
NH2
O
N
CH2 O
N
N
N
O OH
O P O
–
O
N
CH2 O
N
O
NH
N NH2
3´
O OH
O P O
–
O
NH2
N
CH2 O
N
3´
OH OH
O
Freeman’s“Prebiotic Soup”
= The RNA world ?
• Nucleotide monomers
(ATP, GTP, CTP, UTP)
= three phosphate groups
bonded to ribose, which is
bonded to a base.
• In lab simulations they
will form RNA
• And in lab simulations RNA can use the
nucleotide monomers to replicate itself.
Figure 3.15a
RNA basepairing and
hydrogen
bonds
5´
H
Cytosine
N
H
C
Sugar-phosphate backbone
H
C
H
N
C
N
H
H
C
N
N
C
O
C
C
G
N
C
N
H
O
C
C
3´
Guanine
N
H
H Adenine
Uracil
O
C
H
N
N
C
N
H
C
C
A
N
C
C
O
3´
H
N
C
U
C
H
C
N
N
H
5´
Figure 3.16, left
RNA FORMS A TEMPLATE FOR ITS SYNYTHESIS
A
G
U
G
3´
U
C
3´
A
5´
C
5´
1. Complementary
bases pair.
C
G
A
U
G
C
U
G
3´
A
C
C
5´
2. Copied strand polymerizes.
G
A
G
U
C
U
A
G
C
3´
5´
Copied strand
G
3´
Template strand
3´
5´
3´
Copied strand
C
5´
Template strand
Template strand
5´
3. Copy and template separate.
Figure 3.16, right
A
C
5´
4. Copy serves as new
template.
3´
G
A
U
G
C
U
A
G
C
5´
5. New copy polymerizes.
3´
C
G
A
New copy
strand
U
C
5´
U
G
C
U
A
G
C
3´
5´
New
template strand
3´
C
3´
New template strand
5´
G
U
New copy strand
G
3´
5´ A
3´
Copied
strand = new template
C
G
5´
3´
5´
6. New copy is identical
to original template.
What Constitutes Life?
• First living entity was likely some type of
self-replicating macromolecule that could:
– Act as a template.
– Catalyze reactions.
• Could that be a protein?
• Or . . .
The RNA world ?
• RNA can be both catalyst and template:
• Catalytic RNAs occur in some organisms.
• An RNA strand can be a template for forming a
The RNA world ?
The RNA world ?
• A self-replicating RNA could have been the
first living entity.
• Laboratory experiments can generate self-replicating
RNAs.
• Selection of self-replicating RNAs demonstrates
evolution in vitro.
Figure 3.19
HOW RNAs ARE
MADE FOR
SELECTION
EXPERIMENTS
Hairpin loop
3´
C
A
G
Tag sequence U
G
5´
Oligo substrate
5´ G
U
C
A
C
Randomly
generated
sequence
220 nucleotides
3´
Pool RNA
1. Complementary
base pairing
occurs between
oligo substrate
and pool RNA.
5´
3´ CG
AU
GC
UA 220 nucleotides
Tag sequence
GC
5´
3´
2. Ligation occurs
via formation of a
phosphodiester
bond.
Tag sequence
5´
CG
AU
GC
UA 220 nucleotides
GC
3´
Figure 3.20
EXPERIMENTAL SELECTION OF RIBOZYMES
If pool RNA sequence can catalyze
ligation reaction...
GC
UA
CG
AU
CG
Tag
5´
5´ Tag
Oligo
substrate
Oligo
substrate
GC
UA
CG
AU
CG
3´ 220 nucleotides
CG
AU
GC
UA
GC
Tag
5´
…any ligated pool RNA sequences will
adhere to the affinity column and be
saved for further experimentation.
Affinity column
3´ 220 nucleotides
Pool RNA
If pool RNA sequence cannot catalyze
ligation reaction...
C
A
G
U
G
5´ Tag
…unligated pool RNA
sequences will pass
through column and
be thrown away.
220 nucleotides
3´
Pool RNA
5´
G
U
C
A
C
3´
Mutation step in the
The RNA world ?
When new ribozymes are made, some of them
have mutations
Figure 3.20
EXPERIMENTAL SELECTION OF RIBOZYMES
If pool RNA sequence can catalyze
ligation reaction...
GC
UA
CG
AU
CG
Tag
5´
5´ Tag
Oligo
substrate
Oligo
substrate
GC
UA
CG
AU
CG
3´ 220 nucleotides
CG
AU
GC
UA
GC
Tag
5´
…any ligated pool RNA sequences will
adhere to the affinity column and be
saved for further experimentation.
Affinity column
3´ 220 nucleotides
Pool RNA
If pool RNA sequence cannot catalyze
ligation reaction...
C
A
G
U
G
5´ Tag
…unligated pool RNA
sequences will pass
through column and
be thrown away.
220 nucleotides
3´
Pool RNA
5´
G
U
C
A
C
3´
Figure 3.21
101
100
Ligation rate (per hour)
10-1
10-2
10-3
10-4
10-5
10-6
0
1
2
3
4
5
6
Round of experiments
7
8
9
10
Figure 3.22
HOW RNA REPLICASE COULD EVOLVE
First living entity
(RNA replicase)
Ribonucleotides
Exact copies
Mutant
copies
Mutant copies
Different
(mutant)
bases
These copies work at the
same rate as the original.
Most mutants work less
well than the original.
A few mutants work
better than the original.
Few or no copies.
Few copies.
Copies of original RNA replicase
become outnumbered by the
more efficient form.
Many copies–some of these have mutations
that allow replicase to work better yet.
Many copies of most efficient
RNA replicase are made.
The RNA world ?
• Chemical Evolution would be Overtaken by Biological
Evolution
• Natural selection becomes the engine that
drives further evolution.
• The RNA world eventually becomes the
DNA world ?
most important concept:
Enzyme action is determined by enzyme shape; enzyme shape is controlled by
its primary structure (its amino acid sequence), all part of the central dogma of
modern biology:
DNA--->RNA--->protein --> us
(Francis Crick)
BUT sometimes the enzymes are RNA;
then ribozymes control chemical
reactions. They replace activation energy
and may help transfer energy to/from ATP
Help Session
• This week
featuring
Cell structure
• Thursday @ 3pm
Next: MEMBRANES
http://www.queens.edu/faculty/jannr/bio103/helpPages/c04membrane.htm
especially membrane proteins!
And especially the enzymes
and transport proteins
both with active sites
MEMBRANE PROCESS TABLE
Download
from
Friday’s
help
page
QUIZ on
everything
except the
“How”
column
PROCESS
ENERGY SOURCE
HOW IT HAPPENS
DIFFUSION
Random molecular
motion
(passive)
Dissolved substances
become evenly
dispersed = net
movement from areas of
high concentration to
areas of lower
concentration for each
substance
independently.
Molecules move
through membrane
faster when they're
smaller and when the
temperature rises.
Like regular diffusion
except ________(fill it
in)______
Facilitated Diffusion
and Channel-mediated
Diffusion
________(fill it
in)______
Osmosis
________(fill it
in)______
Active Transport
Cell energy sources
(usually ATP)
Endocytosis (active)
(phagocytosis)
Exocytosis
(active)
(secretion)
Cell energy sources
________(fill it
in)______
Like regular diffusion
except ________(fill it
in)______
Dissolved substances
become_______
dispersed
= net movement is often
from areas of _____
concentration to areas of
_________
concentration
Membrane surrounds
and engulfs substances,
usually (maybe always)
attached to receptors on
external face of
membrane________(fill
it in)______
Reverse of endocytosis:
________(fill it
in)______
WHAT MOVES THIS
WAY
Small uncharged
molecules, larger
nonpolar molecules:
Oxygen, ethanol,
steroids
materials which fit
carriers or channels:
Sugar, ions,
________(fill it
in)______
________(fill it
in)______
________(fill it
in)______
________(fill it
in)______
_______(fill it in)______
MEMBRANE PROCESS TABLE
PROCESS
ENERGY SOURCE
HOW IT HAPPENS
Endocytosis (active)
(phagocytosis)
Cell energy sources
Membrane surrounds
and engulfs substances,
usually (maybe always)
attached to receptors on
external face of
membrane________(fill
it in)______
Reverse of endocytosis:
________(fill it
in)______
Exocytosis
(active)
(secretion)
________(fill it
in)______
Do the last two rows on your own
WHAT MOVES THIS
WAY
________(fill it
in)______
_______(fill it in)______
Next: MEMBRANES
http://www.queens.edu/faculty/jannr/bio103/helpPages/c04membrane.htm
especially membrane proteins!
And especially the enzymes
and transport proteins
both with active sites
ACTIVE SITE: The glove and
the “hand” are near-perfect matches
–geometrically
–electrochemically
–for hydrophobicity etc.
–for motions and vibrations
–for van der Walls forces.
ACTIVE SITE: The glove and
the “hand” are near-perfect matches
• THE near-perfect match
depends on exact amino acid
sequence in the active site and
in any part of the enzyme
controlling substrate/active
site access.
ACTIVE SITE: the near-perfect
match depends on exact amino acid sequence
• Mutations can change the amino
acid sequence
• *
• **
• ***
• ****
ACTIVE SITE: its shape can also be
changed reversibly by other factors
• Inhibitors can block the active site
• Inhibitors can pull on another part of the
enzyme and stretch the active site out of
shape
• phosphates and other factors can pull on
another part of the enzyme to pull the
active site into the correct shape
Reversible changes
in protein shape
= The cell’s method of adjusting to its needs
• time for hydrolysis?
– or synthesis?
• Time to take in sugar?
– Or time to dump it out?
• Time to send an electrical signal?
– Or time to restore gradients?
ACTIVE SITE: its shape can also be
changed reversibly by other factors
• Inhibitors and phosphates and hormones and other
factors pull on another part of the enzyme to pull the
active site into or out of the correct shape
• usually reversible because the
factors interact with the enzyme’s
hydrogen and ionic bonds,
not the covalent bonds
ACTIVE SITE: its shape can also be
changed by other factors
• pH changes can change the amino acid
interactions so that the shape is wrong or
right.
• Again, affecting ionic hydrogen bonds,
not covalent bonds
• So is a pH effect permanent?
Figure 2.17
[H3O+]
pH
10-14 14
10-13
Oven cleaner
13
10-12 12
Household bleach
Household ammonia
10-11 11
Basic
Neutral
Acidic
Milk of magnesia
1010
10
10-9
9
10-8
8
10-7
7
10-6
6
Human blood
Pure water
Milk
Urine
10-5
5
Black coffee
10-4
4
10-3
3
10-2
2
Tomatoes
Wine
Vinegar, soft drinks, beer
Lemon juice
10-1
1
10
0
Baking soda
Seawater
Stomach acid
ACTIVE SITE: its shape can also be
changed reversibly by other factors
• pH changes can change the amino acid interactions so that the
shape is wrong or right.
• Why does stomach fluid have a low pH?
• Why does the small intestine have a high
pH?
Figure 3.3b, row 4, left
Each amino acid has a different side chain.
H
H3N+
O
C
H3N+
C
H
H
C
CH2
NH
+NH
H3N+
C
C
–
–
O
O
O
CH2
O
–
CH2
CH2
CH2
CH2
CH2
CH2
NH
+NH
3
C
C
O
+NH
2
NH2
Histidine (H)
His
Lysine (K)
Lys
Basic side chains
Arginine (R)
Arg
Figure 3.3b, row 4, right
Each amino acid has a different side chain.
H
H3N+
H
O
C
H3N+
C
O
C
C
–
–
CH2
O
CH2
C
–
O
CH2
O
C
–
O
Aspartate (D)
Asp
O
Glutamate (E)
Glu
Acidic side chains
O
Table 3.1
TABLE 3.1 HOW AMINO ACIDS INTERACT WITH WATER
In this table, the 20 amino acids are ranked according to how likely
they are to interact with water. The ranking is from least likely to
most likely.
Highly hydrophobic
Least likely
Isoleucine
Valine
Leucine
Phenylalanine
Methionine
Less hydrophobic
Alanine
Glycine
Cysteine
Tryptophan
Tyrosine
Proline
Threonine
Serine
Interaction
with
water
Highly hydrophilic
Histidine
Glutamate
Asparagine
Glutamine
Aspartate
Lysine
Arginine
Most likely
Mutations are not reversible
• mutations change the amino acid sequence.
A different amino acid has a different side
chain.
• Changing a side chain may change the
enzyme’s shape and therefore its action.
• Changing a side chain may change the
transport protein’s shape and therefore its
action
MEMBRANE PROCESS TABLE
Download
from
Friday’s
help
page
QUIZ on
everything
except the
“How”
column
PROCESS
ENERGY SOURCE
HOW IT HAPPENS
DIFFUSION
Random molecular
motion
(passive)
Dissolved substances
become evenly
dispersed = net
movement from areas of
high concentration to
areas of lower
concentration for each
substance
independently.
Molecules move
through membrane
faster when they're
smaller and when the
temperature rises.
Like regular diffusion
except ________(fill it
in)______
Facilitated Diffusion
and Channel-mediated
Diffusion
________(fill it
in)______
Osmosis
________(fill it
in)______
Active Transport
Cell energy sources
(usually ATP)
Endocytosis (active)
(phagocytosis)
Exocytosis
(active)
(secretion)
Cell energy sources
________(fill it
in)______
Like regular diffusion
except ________(fill it
in)______
Dissolved substances
become_______
dispersed
= net movement is often
from areas of _____
concentration to areas of
_________
concentration
Membrane surrounds
and engulfs substances,
usually (maybe always)
attached to receptors on
external face of
membrane________(fill
it in)______
Reverse of endocytosis:
________(fill it
in)______
WHAT MOVES THIS
WAY
Small uncharged
molecules, larger
nonpolar molecules:
Oxygen, ethanol,
steroids
materials which fit
carriers or channels:
Sugar, ions,
________(fill it
in)______
________(fill it
in)______
________(fill it
in)______
________(fill it
in)______
_______(fill it in)______
Chapter 2