Download Basic Molecular Biology (1)

Basic Molecular Biology (1)
Spring, 2007
Guangyi Wang, Ph.D.
[email protected]
Character of Living Organisms
1. Living organisms: Prokaryotes & Eukaryotes
2. Major Differences: Nucleus & Subcellular cytoplasmic
organelles
Internal structure of microbial cells. (a) Diagram of a prokaryote.
(b) Diagram of a eukaryote.
Central Dogma of Life
1. Gene is the element of
information that
specifies the sequence
of amino acids of the
protein.
2. The genetic information
is stored in the
sequence of the DNA
and specify the
sequence of a protein
through an intermediary
4. Steps in genetic information flow
macromolecule (RNA).
(only one of the two strands of 3. Protein is the one that
the DNA double helix is
do the actual functional
transcribed).
work.
Genetic Info Transfer in Prokaryote
1. Operon- a cluster of
genes whose expression
is controlled by a single
operator
2. Messenger RNA
(mRNA)- an RNA
molecule that contains
the genetic information
necessary to encode a
particular protein
3. Prokaryote-a single mRNA often contains more than one
coding region (such mRNAs are called polycistronic).
4. Promoter-a site on DNA to which RNA polymerase can bind
and begin trascription.
Genetic Info Transfer in Eukaryote
1. Exon- the coding DNA sequences
in a split gene (contrast to intron).
2. Intron- the intervening noncoding
DNA sequences in a split gene
(contrast to exon).
3. The mRNAs of eukaryotes are
almost always monocistronic.
4. Each eukaryotic gene has its own
promoter.
Nucleotide, Base, H Bond
Shown is a ribonucleotide, found in RNA.
Deoxyribonucleotides, found in DNA, have an H atom
instead of an OH group on the 29 carbon
Nucleotide, Base, H Bond
Structure of the
bases of DNA and
RNA. Uracil (U) is
not shown, but its
structure is the
same as thymine,
except that it lacks
the methyl group on
C-5
Nucleotide, Base, H Bond
1. Hydrogen bonding. In
nucleic acids, hydrogen
bonds are often
depicted as lines rather
than dots, with two lines
between
adenine/thymine pairs
and three lines between
guanine/cytosine pairs
2. The number of H-bond
is one of key factors to
consider for primer
designing !!!
DNA & RNA
Structure of part of a DNA
chain. The nitrogen bases
can be adenine, guanine,
cytosine, or thymine. In
RNA, an OH group is
present on the 2´ carbon
of the pentose sugar and
uracil replaces thymine.
DNA & RNA
(b) Simplified structure of DNA in which only the nitrogen bases
are shown. The two strands are complementary in base
sequence (A=T; G≡C) and bonded by hydrogen bonds. (c)
Simplified structure of RNA
DNA Replication, Structure & PCR
2
1
2. Linear DNA with complementary singlestranded ends (‘‘sticky ends’’) can cyclize by
base pairing of the complementary ends.
1. Complementary and
antiparallel nature of DNA
DNA Replication, Structure & PCR
1
1. DNA replication is a semiconservative
process in both prokaryotes and eukaryotes
2. Events at the DNA
replication fork. Note the
2
polarity and antiparallel
nature of the DNA
strands. The substrates
for primase are
ribonucleotide
triphosphates, while for
DNA polymerase, they
are deoxyribonucleotide
triphosphates.
DNA Replication, Structure & PCR
The polymerase chain reaction (PCR) :
(a) Target DNA is heated to separate the strands
(denaturing, 94 °C), and a large excess of two
oligonucleotide primers, one complementary
to the target strand and one to the
complementary strand, is added along with
DNA polymerase (annealing, 55 °C).
(b) Primer extension yields a copy of the original
double-stranded DNA (extension, 72 94 °C).
(c) Further heating, primer annealing, and primer
extension yields a second double-stranded
DNA.
(d) The second double-stranded DNA.
(e) Two additional PCR cycles yield 8 and 16
copies, respectively, of the original DNA
sequence.
DNA Replication & PCR
dNTP (deoxythymidine
triphosphate, dTTP;
deoxyadenosine triphosphate,
dATP; deoxyguanosin
trhiphosphate, dGTP; and
deoxycytidine triphosphate,
dCTP)
PCR for amplifying specific DNA sequences. Effect of running 20
PCR cycles on a DNA preparation originally containing 10 copies
of a target gene. the graph is semilogarithmic.
Circular DNA Replication
a
a. In circular DNA (plasmid), bidirectional
replication from an origin leads to the
formation of replication intermediates
resembling the Greek letter theta.
b
b. At an origin of replication that directs
bidirectional replication, two replication
forks must start. Therefore, two leading
strands must be primed, one in each
direction.
Transcription
Structure of the RNA–DNA combination that results at the
initiation of DNA synthesis
Transcription-Overview
Ribonucleoside
triphosphates:ATP,
GTP, UTP, and CTP
Steps in RNA synthesis. The initiation and termination sites are specific
nucleotide sequences on the DNA. The sigma factor allows RNA
polymerase to recognize the initiation site (the promoter). The sigma
factor is released during elongation.
Transcription-Overview
RNA polymerase moves down the DNA chain, causing
temporary opening of the double helix and transcription of one
of the DNA strands. When a termination site is reached, chain
growth stops, and the mRNA and polymerase are released.
Transcription-Promoter
The interaction of RNA polymerase with the promoter
Transcription-Terminators
Inverted repeats in transcribed DNA lead to formation of a
stem-loop structure in the RNA.
Transcription-a Unit
A bacteria ribosomal rRNA
transcription unit:
1) an “rRNA operon.”
2) all bacteria contain such operons
3) the “spacer” between 16S and
23S rRNA genes contains a
tRNA gene.
4) other operons this region may
contain more than one tRNA
gene and often one or more
tRNA genes follow the 5S rRNA
gene,
5) Nonribosomal RNAs are not
found in such units in eukaryotes.
Transcription-RNA processing
An overview of the processing
of the pre-mRNA into mature
mRNA in eukaryotes.
1) adding a cap at the 5’end.
2) removing the introns.
3) clipping of the 3’-end of
the transcript while
adding a poly-A tail.
4) All these steps are
carried out in the
nucleus.
5) The location of the start
and stop codons to be
used during translation.
Translation-Ribosomes
1. the site of protein synthesis
2. Each ribosome is constructed of two subunits.
3. Comparison of prokaryotic and eukaryotic ribosomes
Translation-Protein Synthesis
It is very complicated biological process. You are not
required to know details for this course.
Primary, Secondary and Tertiary
Structure of Protein
a. Primary structure- the precise sequence of
monomeric units in an informational macromolecule.
AASDSLVEHVFIV
b) The initial pattern of folding of a polypeptide, usually
dictated by opportunities for H-bond.
α-helix
ß-Sheet
Primary, Secondary and Tertiary
Structure of Protein
c) Tertiary structure- the final folded structure of a
polypeptide that has previously attained secondary
structure.
Denaturation of Protein
1. Denaturation of the protein
ribonuclease (whose structure
was shown in previous slide).
2. Harsh denaturation yields a
permanently destroyed
molecule (from the standpoint
of biological function) because
of improper folding.
3. This will closely related to
proteomics in the future
lecture.
Summary
• Basic terminology
• Main events of replication, transcription,
and translation.
• Major component of 70S ribosome.
• Protein structure and function.
• General understanding of the genetic
information flow from DNA to protein and
protein to function.