Notesale: Turn your study into money

Document Preview

Extracts from the notes are below, to see the PDF you'll receive please use the links above

---

The Molecular Basis of Inheritance
G

C
A

T

T

A

1 nm
C

G
C
A
C

3
...
7a, c

C

0
...
D
...
H
...
Nature
171 (1953), p
...


James D
...

Crick

Maurice H
...

Wilkins
What about?
Rosalind Franklin

The Structure of DNA
• DNA is composed of four nucleotides,
each containing: adenine, cytosine,
thymine, or guanine
...

• DNA is a double-stranded helix with
antiparallel strands [Watson and Crick]
...


The Basic Principle: Base Pairing to a
Template Strand
• The relationship between structure and
function is manifest in the double helix
• Since the two strands of DNA are
complementary each strand acts as a
template for building a new strand in
replication

DNA replication
• The parent molecule unwinds, and two new
daughter strands are built based on basepairing rules
T

A

T

A

T

A

C

G

C

G

C

T

A

T

A

T

A

A

T

A

T

A

T

G

C

G

C

G

A

T

A

T

A

T

C

G

C

G

C

G

T

A

T

A

T

A

T

A

T

A

T

C

G

C

G

C

C

G

A
G

(a) The parent molecule has two
complementary strands of DNA
...


(b) The first step in replication is
separation of the two DNA
strands
...


(d) The nucleotides are connected
to form the sugar-phosphate
backbones of the new strands
...


DNA Replication is “Semi-conservative”
• Each 2-stranded
daughter molecule is
only half new
• One original strand was
used as a template to
make the new strand

DNA Replication
• The copying of DNA is remarkable in its speed and
accuracy
• Involves unwinding the double helix and synthesizing
two new strands
...


Parental (template) strand
Daughter (new) strand

Bubble

0
...

3 Eventually, the replication
bubbles fuse, and synthesis of
the daughter strands is
complete
...


(b) In this micrograph, three replication
bubbles are visible along the DNA of
a cultured Chinese hamster cell (TEM)
...


The Mechanism of DNA Replication
• DNA synthesis on the leading strand is continuous
• The lagging strand grows the same general direction as the leading
strand (in the same direction as the Replication Fork)
...


•

DNA polymerase III extends the strand in the
5’-to-3’ direction

•

DNA polymerase I degrades the RNA primer
and replaces it with DNA

•

DNA ligase joins the DNA fragments into a
continuous daughter strand

Enzymes in DNA replication

Helicase unwinds
parental double helix

DNA polymerase III
binds nucleotides
to form new strands

Binding proteins
stabilize separate
strands

Primase adds
short primer
to template strand

DNA polymerase I
(Exonuclease) removes
RNA primer and inserts
the correct bases

Ligase joins Okazaki
fragments and seals
other nicks in sugarphosphate backbone

Replication
3’
3’

5’

5’
3’
5’

3’

5’

Helicase protein binds to DNA sequences called
origins and unwinds DNA strands
...

Primase protein makes a short segment of RNA
complementary to the DNA, a primer
...


Replication
Overall direction
of replication

3’
5’

3’

5’

3’
5’

3’
5’

DNA polymerase enzyme adds DNA nucleotides
to the RNA primer
...


Replication
Overall direction
of replication

3’

3’

5’

5’
3’
5’

3’
5’

Leading strand synthesis continues in a
5’ to 3’ direction
...


Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments
...

Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments
...

Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments
...

Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments
...


Replication
3’
3’
5’
3’

5’

3’5’

3’
5’

Polymerase activity of DNA polymerase I fills the gaps
...


Replication Fork Overview

Proofreading
• DNA must be faithfully replicated…but
mistakes occur
– DNA polymerase (DNA pol) inserts the wrong
nucleotide base in 1/10,000 bases
• DNA pol has a proofreading capability and can correct
errors

– Mismatch repair: ‘wrong’ inserted base can be
removed
– Excision repair: DNA may be damaged by
chemicals, radiation, etc
...

• DNA polymerase proofreads and repairs
accidental mismatched pairs
...
Each of
us probably inherited 3-4 mutations!

Proofreading and Repairing DNA
• DNA polymerases
proofread newly made
DNA, replacing any
incorrect nucleotides
• In mismatch repair of DNA,
repair enzymes correct
errors in base pairing
• In nucleotide excision DNA
repair nucleases cut out
and replace damaged
stretches of DNA

1 A thymine dimer
distorts the DNA molecule
...

Nuclease

DNA
polymerase

3 Repair synthesis by
a DNA polymerase
fills in the missing
nucleotides
...


Accuracy of DNA Replication
• The chromosome of E
...


– Takes a cell a few hours to copy this DNA
– With amazing accuracy – an average of 1 per
billion nucleotides

Protein Synthesis
• The information content of DNA is in the form of
specific sequences of nucleotides along the DNA
strands
• The DNA inherited by an organism leads to specific
traits by dictating the synthesis of proteins
• The process by which DNA directs protein synthesis,
gene expression includes two stages, called
transcription and translation

Transcription and Translation
• Cells are governed by a cellular chain of command
– DNA RNA protein

• Transcription
– Is the synthesis of RNA under the direction of DNA
– Produces messenger RNA (mRNA)

• Translation
– Is the actual synthesis of a polypeptide, which occurs under
the direction of mRNA
– Occurs on ribosomes

Transcription and Translation
• In a eukaryotic cell the nuclear envelope separates transcription
from translation
• Extensive RNA processing occurs in the nucleus

Nuclear
envelope

DNA

TRANSCRIPTION

Pre-mRNA

RNA PROCESSING

mRNA

Ribosome

TRANSLATION
Polypeptide

(b) Eukaryotic cell
...
The original RNA
transcript, called pre-mRNA, is processed in various
ways before leaving the nucleus as mRNA
...


Table 17
...
After RNA polymerase binds to
the promoter, the DNA strands unwind, and
the polymerase initiates RNA synthesis at the
start point on the template strand
...
The polymerase moves downstream, unwinding the
DNA and elongating the RNA transcript 5  3 
...

Rewound
RNA

RNA
5
3

3
5

3
5

RNA

transcript

3 Termination
...


5
3

3
5
5

Completed RNA
transcript

3

Synthesis of an RNA Transcript - Initiation
1 Eukaryotic promoters

TRANSCRIPTION
RNA PROCESSING

• Promoters signal the initiation
of RNA synthesis
• Transcription factors help
eukaryotic RNA polymerase
recognize promoter sequences

DNA

Pre-mRNA

mRNA
TRANSLATION

Ribosome
Polypeptide

Promoter

5
3

3
5

T A T A A AA
AT AT T T T

TATA box

Start point

Template
DNA strand

Several transcription
factors

2

Transcription
factors

5
3

3
5
3 Additional transcription

factors

RNA polymerase II

5
3

Transcription factors

3
5

5
RNA transcript
Transcription initiation complex

Synthesis of an RNA Transcript - Elongation
•

RNA polymerase synthesizes a single strand of RNA against the DNA template strand (antisense strand), adding nucleotides to the 3’ end of the RNA chain

•

As RNA polymerase moves along the DNA it continues to untwist the double helix, exposing
about 10 to 20 DNA bases at a time for pairing with RNA nucleotides

Non-template
strand of DNA

Elongation

RNA nucleotides
RNA
polymerase

A

3

T

C

C

A

A

3 end
U
A

E

G

C

A

T

5

A

G

G

T

T

Direction of transcription
(“downstream”)

5

Newly made
RNA

Template
strand of DNA

Synthesis of an RNA Transcript - Termination
•
•

Specific sequences in the DNA signal termination
of transcription
When one of these is encountered by the
polymerase, the RNA transcript is released from
the DNA and the double helix can zip up again
...
mRNA requires processing
...
After this, the messenger RNA
moves to the cytoplasm for translation
...
These both
function to protect the RNA from enzymes that would degrade

•

Most of the genome consists of non-coding regions called introns
–
–

•

Non-coding regions may have specific chromosomal functions or have regulatory purposes
Introns also allow for alternative RNA splicing

Thus, an RNA copy of a gene is converted into messenger RNA by doing 2 things:
–

Add protective bases to the ends

–

Cut out the introns

Alteration of mRNA Ends
• Each end of a pre-mRNA molecule is modified in a
particular way
– The 5 end receives a modified nucleotide cap
– The 3 end gets a poly-A tail

A modified guanine nucleotide
added to the 5 end
TRANSCRIPTION

RNA PROCESSING

50 to 250 adenine nucleotides
added to the 3 end

DNA

Pre-mRNA

5
mRNA

Protein-coding segment

Polyadenylation signal
3

G P P P

AAUAAA

AAA…AAA

Ribosome
TRANSLATION

5 Cap
Polypeptide

5 UTR

Start codon Stop codon

3 UTR

Poly-A tail

RNA Processing - Splicing
• The original transcript from
the DNA is called pre-mRNA
...


• The introns are removed by a
process called splicing to
produce messenger RNA
(mRNA)

RNA Processing
• Proteins often have a modular architecture consisting
of discrete structural and functional regions called
domains
• In many cases different exons code for the different
domains in a protein
Gene

DNA
Exon 1

Exon 2

Intron

Intron

Exon 3

Transcription
RNA processing
Translation
Domain 3
Domain 2
Domain 1

Figure 17
...

tRNA brings the amino acids to the ribosomes,

3

A
C
C
A 5
C G
The “anticodon” is the 3 RNA bases that
G C
C G
matches the 3 bases of the codon on the
U G
U A
mRNA molecule
A U
A U
U C
UA
C A C AG
*
G
*
G U G U *
C
C
* *
U C
*
* G AG C
(a) Two-dimensional structure
...
The anticodon triplet is
A
A*
C
unique to each tRNA type
...
)
A
G
A

Amino acid
attachment site

Anticodon

C U C
G A G

A G *
*
G
A G G

Hydrogen
bonds

Transfer RNA
• 3 dimensional tRNA molecule is roughly “L” shaped

5
3

Amino acid
attachment site

Hydrogen
bonds

A AG
3
Anticodon
(b) Three-dimensional structure

5

Anticodon

(c) Symbol used
in the book

Ribosomes
•
•

Ribosomes facilitate the specific coupling of tRNA anticodons with
mRNA codons during protein synthesis
The 2 ribosomal subunits are constructed of proteins and RNA
molecules named ribosomal RNA or rRNA

DNA

TRANSCRIPTION

mRNA
Ribosome
TRANSLATION

Polypeptide

Growing
polypeptide

Exit tunnel

tRNA
molecules

Large
subunit
E

P A
Small
subunit

5
mRNA

3

(a) Computer model of functioning ribosome
...
The eukaryotic ribosome is roughly
similar
...


Building a Polypeptide
Amino end

Growing polypeptide

Next amino acid
to be added to
polypeptide chain

tRNA
3

mRNA

5

Codons

(c) Schematic model with mRNA and tRNA
...
The P site holds the tRNA attached to the growing polypeptide
...
Discharged
tRNA leaves via the E site
...
In a prokaryotic cell, the mRNA binding site
on this subunit recognizes a specific nucleotide
sequence on the mRNA just upstream of the start
codon
...
This tRNA
carries the amino acid methionine (Met)
...
Proteins called initiation
factors (not shown) are required to bring all the
translation components together
...
The initiator tRNA is
in the P site; the A site is available to the tRNA
bearing the next amino acid
...
The anticodon
of an incoming aminoacyl tRNA
base-pairs with the complementary
mRNA codon in the A site
...


Amino end
of polypeptide

DNA
mRNA
Ribosome

TRANSLATION

Polypeptide

E
mRNA
Ribosome ready for
next aminoacyl tRNA

5

3
P A
site site

2 GTP
2 GDP

E

E
P

P

A

GDP

3 Translocation
...
The empty tRNA
in the P site is moved to the E site,
where it is released
...


GTP

E
P

A

A
2 Peptide bond formation
...
This step
attaches the polypeptide to the
tRNA in the A site
...

a release factor instead of tRNA
...

The polypeptide is thus freed
from the ribosome
...
When the
ribosome reaches a STOP codon, there is no
corresponding transfer RNA
...

• The release factor causes the whole complex to fall
apart: messenger RNA, the two ribosome subunits,
the new polypeptide
...


A summary of transcription and translation in a eukaryotic cell
DNA

TRANSCRIPTION
1
RNA is transcribed

from a DNA template
...


RNA transcript
(pre-mRNA)
Intron
Aminoacyl-tRNA
synthetase
NUCLEUS
Amino
acid

FORMATION OF
INITIATION COMPLEX

CYTOPLASM

AMINO ACID ACTIVATION

tRNA

3 After leaving the

4

Each amino acid
attaches to its proper tRNA
with the help of a specific
enzyme and ATP
...

mRNA

Growing
polypeptide
Activated
amino acid

Ribosomal
subunits

5
TRANSLATION
5

E

A

A A A
U G G U U U A U G

Figure 17
...

(When completed, the
polypeptide is released
from the ribosome
...

• It might also be inserted into a membrane, if
translated by a ribosome bound to the
endoplasmic reticulum
...

• Sometimes other molecules are also attached
to the polypeptides: sugars, lipids,
phosphates, etc
...


Mutation Causes and Rate
• The natural replication of DNA produces occasional
errors
...
\
• Typically genes incur base substitutions about once
in every 10,000 to 1,000,000 cells
...

• However, most mutations are neutral: have no effect
...

• Mutations in other body cells only cause trouble when
they cause cancer or related diseases
...
Generally, only one or a few
base pairs are involved
...

Physical agents include high-energy radiation like X-rays and
ultraviolet light
Chemical mutagens fall into several categories
...

Interference with DNA replication by inserting into DNA and distorting the
double helix
...


Tests are often used as a preliminary screen of chemicals to identify
those that may cause cancer
Most carcinogens are mutagenic and most mutagens are carcinogenic
...


Point Mutation
• The change of a single nucleotide in the DNA’s
template strand leads to the production of an
abnormal protein
Wild-type hemoglobin DNA
3

Mutant hemoglobin DNA

C T

T

G U A

5

In the DNA, the
mutant template
strand has an A where
the wild-type template
has a T
...


3

5

T

C A

mRNA

mRNA
G A

A

5

3

5

3

Normal hemoglobin

Sickle-cell hemoglobin

Glu

Val

The mutant (sickle-cell)
hemoglobin has a valine
(Val) instead of a glutamic
acid (Glu)
...

Wild type
mRNA
Protein

5

A U GA A GU U U G G C U A A
Met

Lys

Gly

Phe

Stop

Amino end
Carboxyl end
Base-pair insertion or deletion
Frameshift causing immediate nonsense

Extra U
AU G U A AG U U U G GC U A
Met

Stop

Frameshift causing
extensive missense

U Missing

A U G A A GU U G G C U A A
Met

Lys

Leu

Ala

Insertion or deletion of 3 nucleotides:
no frameshift but extra or missing amino acid

A AG

Missing

A U G U U U G G C U A A

Met

Phe

Gly

Stop

3



---