Nucleic Acid Structure:
Purine and
Pyrimidine nucleotides can be combined to form
nucleic acids:
1. Deoxyribonucliec acid (DNA) is composed of deoxyribonucleosides of
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Adenine
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Guanine
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Cytosine
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Thymine
2. Ribonucleic acid (RNA) is composed of ribonucleoside of
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Adenine
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Guanine
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Cytosine
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Uracil
Purines and pyrimidines are
critical bc of their use in:
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the synthesis of ATP
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cofactors
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RNA
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DNA and other important cell
components
Nearly all mos can
synthesize their own purines and pyrimidines – they are critical to cell
function.
Purines and Pyrimidines are
cyclic nitrogenous bases with several double bonds and aromatic properties.
Purines:
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Two joined rings
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Adenine and guanine are
commonly found in MOs
Pyrimidines:
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One ring
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Uracil, cytosine and
thymine are commonly found in MOs
A purine or pyrimidine base
joined with a pentose sugar, either ribode or deoxdyribose is a nucleoside.
Nucleotide: is a nucleoside with one or
more phosphate groups attached to a sugar.
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In both DNA and RNA,
nucleosides are joined by phosphate groups to form long polynucleotodes chains.
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Difference in chemical
composition is the:
Sugar and pyrimidines bases:
DNA: deoxyribose and thymine
RNA: ribose and uracil in place of thymine
Structure and
function of DNA:
1. Bacterial
chromosomes consist of single circular molecules of double-stranded DNA
(dsDNA).
a. Size of the E. coli
chromosome is 4.7 x 106 base pairs
(bp).
b. Size of an average bacterial
gene is 1,000 bp (E. coli has about 3,500 genes vs. human 30,000).
2. Structure of DNA
a. Two polynucleotide chains
that contain the bases adenine (A), cytosine (C), guanine (G), and thymine (T).
b. DNA is a double helix
consisting of complementary strands
the bases in one strand
match up with those of other according to the base pair rules.
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purine Adenine (A) is always
paired with the pyrimidine thymine (T)
§
A = T
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Purine guanine (G) is always
paired with the pyrimidine
Cytosine.
§
G = C, hydrogen bonds broken
by heat).
c. Two properties of the genetic
material.
(1) The genetic material
undergoes replication prior to cell division.
(2) The genetic material
directs protein synthesis.
d. Important concept: a single nucleic acid strand (usually DNA)
can be used to guide the synthesis of a complementary strand
(either DNA in replication or RNA prior to protein synthesis). The original strand is a template for
complementary strand synthesis.
3. RNA Structure:
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Usually single stranded
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Can coil back upon itself to
form a hairpin-shaped structure with complementary base pairing and helical
organization
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Cells contain three (3)
different types of RNA:
1.
messenger RNA – mRNA
2.
ribosomal RNA – rRNA
3.
transfer RNA – tRNA
(differ in
function,, site of synthesis in ec, and structure)
DNA replication:
1. Bidirectional Replication
a. Replication starts at a
single site on the circular DNA E. coli chromosome (origin of replication).
b. Replication stops at a site
about half-way around the chromosome (the terminus
of replication).
c. Replication forks are the sites of new DNA synthesis,
the place where the DNA helix is unwound and individual strands are replicated.
d. Replication of the E. coli
chromosome is bidirectional (two
replication forks moving in opposite directions around the chromosome).
e. Replication continues until
the entire replicon is replicated
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Replicon: portion of the genome that contains an orgin and is replicated as
a unit.
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And when the replication
forks move around the circle, the bacterial chromosome is a single replicon,
the forks meet on the other side and
two separate chromosomes are released.
2. Rolling Circle Replication
§
Occurs during E.coli
conjugation and reproduction of viruses.
a. One strand of DNA is nicked and the free 3’
hydroxyl end is extended
by replication enzymes.
b. As 3’
end is lengthened while the growing point rolls around the
circular template, the 5’ end of strand is displaced and forms
an
everlengthening tail.
c. The single stranded tail may be converted to
the double stranded form
by complementary strand synthesis.
d. Useful for viruses bc allows rapid and
continuous production of many
genomes copies from a single initiation
event.
Mechanism of DNA Replication:
(1) The double helix is unwound by DNA helicase (requires ATP for activity).
(2) Single-strand
DNA binding protein
(SSBs) binds to and stabilizes the
unwound DNA strands.
(3) During the process of rapid unwinding the DNA, tension,
supercoils and supertwists can occur in
the helix, tension relieved and continued unwinding
is promoted by the a topoisomerase.
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Topoisomerase changes the
structure of DNA by breaking one or two strands that it remains unaltered as
its shape is changed.
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DNA gyrase is an E.coli
topooisomerase.
(3) DNA polymerase III
requires a primer (a free 3'
end onto which new nucleotides can
be attached). Therefore, short RNA
primers are first synthesized by primase.
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DNA polymerase enzymes: catalyzes the synthesis of DNA in the 5’ to 3’ direction and
reading the DNA template in the 3’ to 5’ direction.
(4) DNA polymerase
III synthesizes the new complementary DNA strands using each parental strand as a template.
(a)
Synthesis is continuous on
one strand and discontinuous on the other.
- DNA replication always proceed from the 5’
phosphate to the
3’hydroxyl.
-
Leading Strand: strand growing from the 5’
phosphate to the
3’
hydroxyl.
- DNA synthesis can occur continuously bc
there is always a free 3’OH at the
replication fork to which a new nucleotide
can be added.
- Lagging
Strand: the opposite strand, DNA
synthesis must occur
discontinuously (bc there is
no 3’OH at the
replication fork to which a
new nucleotide can
attach.)
(b) Discontinuous fragments (Okazaki fragments) are about 1,000 nucleotides
in length.
(5) DNA polymerase
I removes the RNA primers and replaces them with DNA (repair enzyme).
(6) DNA ligase
joins and seals the pieces of newly-synthesized DNA together.
f. The enzymes involved in DNA
replication form a complex (the replisome) at each replication fork.
g. Both of the daughter
chromosomes consist of one parental DNA strand and one newly-synthesized DNA
strand (semi-conservative replication).
a.
The sequence of bases along the double helix can be read by cell
machinery and used as a blueprint to make proteins (the genetic code Table11.5).
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DNA base sequence corresponds to the amino acid sequence of the
polypeptide specified by the gene.
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Mutations are the results of changes of single amino acids in a
polypeptide chain.
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There are 20 amino acids present in a protein, therefore there must be
20 different code words in a linear single strand of DNA.
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Codons: code words, a sequence of
three bases in messenger RNA that encodes for specific amino acid.
1.
Code degeneracy: there are up
to six different codons for a given amino acid.
2.
61 codons, Sense codons – direct amino acid incorporation into
protein.
3.
Stop or Nonsense codons: the
remaining 3 (UGA, UAG, UAA) are involved in the termination of
translation.
b. The genetic information encoded in DNA directs protein
synthesis in two steps: transcription
and translation.
Transcription: synthesis of RNA under the
direction of DNA
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Generates three (3) KINDS OF
RNAs
(1). Messenger (mRNA):
o
bears the message for
protein synthesis
(2). Transfer (tRNA):
o
carries amino acids during
protein synthesis
(3). Ribosomal (rRNA):
o
molecules are components of
ribosomes
(1) Gene: a sequence of bases in DNA that specifies a
single polypeptide (or, in some cases, a single RNA molecule rRNA, tRNA).
(2) Organization of a typical gene in E. coli: promoter - coding
region - terminator.
(3) RNA polymerase
enzyme makes a single-stranded RNA copy (messenger RNA) that is
complementary to one of the DNA strands (the template strand) of the gene, therefore mRNA is
synthesized under the direction of DNA.
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Like DNA, RNA synthesis processed in the 5’ to 3’ direction with new
nucleotides being added to the 3’ end of the growing chain.
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There are two enzymes in E.coli that help aid in the transcription
process:
1. Core
Enzyme: helps by catalytic RNA synthesis and contains four types of polypeptide chain.
2. Sigma
Factor Enzyme: has no catalytic activity but helps the core enzyme recognize the start genes. Once
RNA synthesis begins, the sigma factor
dissociates from the core enzyme-DNA complex and is available to aid another core enzyme
(a) RNA polymerase recognizes a specific base sequence with the aid
of the sigma factor and binds to the promoter,
where it unwinds the DNA strands (16 base pairs) and begins mRNA synthesis (on
one of the DNA strands - the coding strand).
(b) Enzyme continues ATP-dependent mRNA synthesis as it moves
through the coding region.
(c) Enzyme stops at the terminator
and releases both mRNA and DNA.
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Terminators: stop signals to mark the end of a gene or sequence of genes and
stop transcription by the RNA polymerase.
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Two kinds of terminators:
1.
Stretch
of six uridine residues following the
mRNA
and causes the polymerase to stop
transcription
and release the mRNA without
the aid
of any accessory factors.
2. Rho Factor: Special protein
It is thought that the rho binds to mRNA and
moves
alongthe molecule until it reaches the
RNA
polymerase that has halted at a terminator.
Rho then
causes the polymerase to dissociate
from the
mRMA, probably by unwinding the
mRNA-DNA complex.
Translation or Protein Synthesis:
Translation: mRNA nucleotide sequence is translated into
the amino acid sequence of a polypeptide chain.
(1) Each set of three bases in mRNA (a codon) specifies one
amino acid in a protein.
(2) The “dictionary” of codons constitutes the genetic code
(3) Amino acids do not line up directly with the codons: an adapter molecule called transfer RNA is required.
(a) The amino acid is attached to the 3’ end of the tRNA molecule.
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The 3’ end of all tRNAs has the same C-C-A sequence.
(b) Other end of the tRNA molecule carries a triplet of bases (the anticodon)
that is complementary to the mRNA codon.
(c). The overall structure of the tRNA is a cloverleaf structure p. 266
(d). Amino acids are activated
for protein synthesis through a reaction catalyzed by aminoacyl-tRNA synthetases
AA + tRNA + ATPŕ aminoacyl-tRNA = AMP + ppi
Process
where the amino acid is activated:
-- amino acid is attached to the 3’ OH of the tRNA and readily transferred to the
end of the growing polypeptide chain.
(4) Amino acid-charged tRNA’s and mRNA are brought together at a
complex cellular organelle called a ribosome.
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The place where protein synthesis actually takes place
The ribosome consists of two subunits.
(a) small (30S) subunit: 1 ribosomal RNA molecule and
21 proteins
(b) large (50S) subunit: 2 ribosomal RNA molecules and 32 proteins
Steps in Translation (Protein Synthesis)
(a) Initiation of
polypeptide synthesis when mRNA, small ribosomal subunit, first charged tRNA,
and accessory initiation factors bind together (initiator codon AUG or GUG).
Binding of the large ribosomal subunit follows.
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Bacteria begin translation with a N-formymethionyl-tRNAfMet
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Eucaryotic and Archeal (except mitochindrian and chloroplast) begin
translation with a special initiator methinoyl-tRNAMet
(b) Elongation of
the polypeptide occurs as new tRNA's are brought in and old tRNA's are expelled
from the ribosome following peptide bond synthesis.