Chapter 5
Macromolecules and Molecular Genetics
?The key informational macromolecules in
prokaryotic and eukaryotic cells are DNA,
RNA,and protein.
?Molecular Genetics deals with the
mechanisms by which the information in
DNA is replicated,and by which it can be
transcribed into RNA and translated into
specific proteins.
Central Dogma of Molecular Biology
中心法则
One-way transfer
of genetic information
from nucleic acid
to protein is call
Central Dogma of
Molecular Biology
Information Transfer in
Prokaryotes and Eukaryotes
Prokaryotes
Eukaryotes
DNA Synthesis proceeds 5’ 3’
A,Adenine
G,Guanine
T,Thymine
C,Cytosine
Replication of DNA proceeds by insertion of a new
nucleoside triphosphate at the free 3’-hydroxyl end,with
loss of two phosphate
DNA double
helix
Many
important
proteins
interact
with the
major
groove of
DNA
double
helix
How DNA is supercoilded and relaxed
?Enzyme involved in supercoiling (in
bacteria):
– DNA gyrase (旋转酶）：
? DNA Topoisomerase II (DNA拓朴异构酶 II)
?Enzyme involved in removing supercoiling:
– DNA gyrase:
? DNA Topoisomerase I
?Two antibiotics (Nalidixic acid and
Novobiocin) inhibit the action of DNA.
Secondary Structure of DNA
?DNA bending,involving several runs of five to six
adenines (in the same strand),each separated by four
or five bases;
?Inverted repeat,regions of DNA containing
repeated sequences in inverse orientation,leading to a
stem-loop (茎 — 环） ;
?Sticky end,DNA with single-strand complementary
sequences at the end,this can form a hairpin,linear
DNA can form a cycle with their sticky end.
?These structures influence DNA interaction
with proteins
The effect of temperature on
DNA structure
?Two hydrogen bonds hold adenine-thymine (A-T)
together;
?Three hydrogen bonds hold guanine-cytosine (G-
C) together;
?Thus,G-C pairs are stronger than A-T pairs;
?The separation of double stranded DNA by
heating is called melting;
?DNA with higher GC content has higher melting
temperature;
?Organisms living at high T has higher GC content.
Hybridization of nucleic acids
?Hybridization,complementary base pairing;
?Forms,DNA:DNA,DNA:RNA;
?The application of a radioactive PROBE to
detect a complementary DNA fragment;
?Southern blot procedure,DNA in gel,RNA
or DNA is used as a probe;
?Northern blot procedure,RNA in gel,DNA
or RNA is used as a probe.
Interaction of proteins with nucleic acids
Central to replication,transcription and translation
Interaction of proteins with nucleic acids
Central to the regulation of replication,
transcription and translation processes
?Protein recognizes contact points that are
associated with specific base sequences in
DNA;
?Inverted repeats are frequently the locations
at which protein molecules combine
specifically with DNA
Nucleotide sequence of the operator gene of the lactose operon
Working with Nucleic acids,The tools
1,Extraction and purification of DNA (lysozyme or detergent
plus alcohol precipitation);
2,UV detection of the presence of DNA at 260 nm;
3,CsCl Density gradient centrifugation of DNA;
4,Gel electrophoresis
5,Detecting DNA by fluorescence (Ethidium Bromide,溴化
乙锭 )；
6,Radioactive labelling (autoradiography);
7,Effect of temperature on nucleic acids (detect GC content);
8,Nucleic acid hybridization (Southern blot,Northern blot);
9,Determine the DNA sequences (radio or fluorescent
labelling).
Restriction Enzymes
?Restriction endonucleases are used by organisms to
destroy invasion of foreign DNA,they are specific
in their action;
?Many of these DNA sequences exhibit twofold
symmetry around a given DNA point;
?The two DNA strands are palindrome;
?The endonucleases break double-stranded DNA,
thus make the broken DNA not repairable;
?Cells protect themselves by chemical modification
(methylation) of DNA to avoid endonuclease cut.
Macromolecules and Molecular Genetics
?The key informational macromolecules in
prokaryotic and eukaryotic cells are DNA,
RNA,and protein.
?Molecular Genetics deals with the
mechanisms by which the information in
DNA is replicated,and by which it can be
transcribed into RNA and translated into
specific proteins.
Central Dogma of Molecular Biology
中心法则
One-way transfer
of genetic information
from nucleic acid
to protein is call
Central Dogma of
Molecular Biology
Information Transfer in
Prokaryotes and Eukaryotes
Prokaryotes
Eukaryotes
DNA Synthesis proceeds 5’ 3’
A,Adenine
G,Guanine
T,Thymine
C,Cytosine
Replication of DNA proceeds by insertion of a new
nucleoside triphosphate at the free 3’-hydroxyl end,with
loss of two phosphate
DNA double
helix
Many
important
proteins
interact
with the
major
groove of
DNA
double
helix
How DNA is supercoilded and relaxed
?Enzyme involved in supercoiling (in
bacteria):
– DNA gyrase (旋转酶）：
? DNA Topoisomerase II (DNA拓朴异构酶 II)
?Enzyme involved in removing supercoiling:
– DNA gyrase:
? DNA Topoisomerase I
?Two antibiotics (Nalidixic acid and
Novobiocin) inhibit the action of DNA.
Secondary Structure of DNA
?DNA bending,involving several runs of five to six
adenines (in the same strand),each separated by four
or five bases;
?Inverted repeat,regions of DNA containing
repeated sequences in inverse orientation,leading to a
stem-loop (茎 — 环） ;
?Sticky end,DNA with single-strand complementary
sequences at the end,this can form a hairpin,linear
DNA can form a cycle with their sticky end.
?These structures influence DNA interaction
with proteins
The effect of temperature on
DNA structure
?Two hydrogen bonds hold adenine-thymine (A-T)
together;
?Three hydrogen bonds hold guanine-cytosine (G-
C) together;
?Thus,G-C pairs are stronger than A-T pairs;
?The separation of double stranded DNA by
heating is called melting;
?DNA with higher GC content has higher melting
temperature;
?Organisms living at high T has higher GC content.
Hybridization of nucleic acids
?Hybridization,complementary base pairing;
?Forms,DNA:DNA,DNA:RNA;
?The application of a radioactive PROBE to
detect a complementary DNA fragment;
?Southern blot procedure,DNA in gel,RNA
or DNA is used as a probe;
?Northern blot procedure,RNA in gel,DNA
or RNA is used as a probe.
Interaction of proteins with nucleic acids
Central to replication,transcription and translation
Interaction of proteins with nucleic acids
Central to the regulation of replication,
transcription and translation processes
?Protein recognizes contact points that are
associated with specific base sequences in
DNA;
?Inverted repeats are frequently the locations
at which protein molecules combine
specifically with DNA
Nucleotide sequence of the operator gene of the lactose operon
Working with Nucleic acids,The tools
1,Extraction and purification of DNA (lysozyme or detergent
plus alcohol precipitation);
2,UV detection of the presence of DNA at 260 nm;
3,CsCl Density gradient centrifugation of DNA;
4,Gel electrophoresis
5,Detecting DNA by fluorescence (Ethidium Bromide,溴化
乙锭 )；
6,Radioactive labelling (autoradiography);
7,Effect of temperature on nucleic acids (detect GC content);
8,Nucleic acid hybridization (Southern blot,Northern blot);
9,Determine the DNA sequences (radio or fluorescent
labelling).
Restriction Enzymes
?Restriction endonucleases are used by organisms to
destroy invasion of foreign DNA,they are specific
in their action;
?Many of these DNA sequences exhibit twofold
symmetry around a given DNA point;
?The two DNA strands are palindrome;
?The endonucleases break double-stranded DNA,
thus make the broken DNA not repairable;
?Cells protect themselves by chemical modification
(methylation) of DNA to avoid endonuclease cut.
