Download Nucleic Acid Structure:

Nucleic Acid Structure:
Purine and Pyrimidine nucleotides can be combined to form nucleic acids:
1.
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Deoxyribonucliec acid (DNA) is composed of deoxyribonucleosides
of
Adenine
Guanine
Cytosine
Thymine
!
!
!
!
Ribonucleic acid (RNA) is composed of ribonucleoside of
Adenine
Guanine
Cytosine
Uracil
2.
Purines and pyrimidines are critical bc of their use in:
! the synthesis of ATP
! cofactors
! RNA
! 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:
! Two joined rings
! Adenine and guanine are commonly found in MOs
Pyrimidines:
! One ring
! 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.
! In both DNA and RNA, nucleosides are joined by phosphate groups to
form long polynucleotodes chains.
! 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.
2.
Bacterial chromosomes consist of single circular molecules of doublestranded 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).
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.
! purine Adenine (A) is always paired with the pyrimidine
thymine (T)
! A=T
! 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)
d.
3.
The genetic material directs protein synthesis.
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.
RNA Structure:
! Usually single stranded
! Can coil back upon itself to form a hairpin-shaped structure
with complementary base pairing and helical organization
! 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
! Replicon: portion of the genome that contains an orgin and is
replicated as a unit.
! 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.
! Topoisomerase changes the structure of DNA by breaking one or two
strands that it remains unaltered as its shape is changed.
! 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.
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.
!
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.)
(4)
(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).
Expression of the genetic information
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).
! DNA base sequence corresponds to the amino acid sequence
of the polypeptide specified by the gene.
! Mutations are the results of changes of single amino acids in
a polypeptide chain.
! There are 20 amino acids present in a protein, therefore there
must be 20 different code words in a linear single strand of
DNA.
! 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.
DNA Transcription or RNA Synthesis:
Transcription: synthesis of RNA under the direction of DNA
! 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.
! Like DNA, RNA synthesis processed in the 5’ to 3’ direction with new
nucleotides being added to the 3’ end of the growing chain.
! 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.
! Terminators: stop signals to mark the end of a
gene or sequence of genes and stop
transcription by the RNA polymerase.
! 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.
! 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 + ATP# 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.
! 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.
! Bacteria begin translation with a N-formymethionyltRNAfMet
! 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.
[1]
Initial entry of charged tRNA and peptide bond
formation occur in the A site.
! Aminoacyl or acceptor site
[2]
Expulsion of preceding (now uncharged) tRNA
from the P site.
! Peptidyl or donor site
[3]
Translocation: the final stage in elongation.
Three things happen here:
! Transfer of tRNA-linked polypeptide moves
from the A site into the P site.
! Ribosomes moves one codon along the mRNA
so tha a new codon is positioned in the A site.
! Empty tRNA leaves the P site t the E site and
then leaves the ribosome.
(c)
Release or Termination of polypeptide and mRNA
from the ribosome upon the completion of translation.
! Small and large ribosomal subunits dissociate with
the aid of accessory release factors.
!
Protein synthesis stops when it reaches one of the
nonsense codons:
UAA, UAG, and UGA.
! Release Factors(RF-1, RF-2, RF-3).
aids the ribosome in recognizing the nonsense
codons.
5.
Regulation of gene expression
a.
Expression of most genes can be turned off and on, usually by
controlling the initiation of transcription.
b.
Lactose degradation in E. coli
(1)
LacY protein transports lactose into the cell.
(2)
LacZ protein (β-galactosidase) cleaves lactose (a
disaccharide) into glucose and galactose.
(3)
LacA protein is a nonessential enzyme.
c.
Operon: several genes are transcribed as a single unit, usually
encode proteins involved in a common process (not common in
eukaryotes).
d.
Organization of the lac operon
e.
When lactose is added to the culture medium, transcription of the
lac operon is induced. In the absence of lactose, transcription is
repressed.
f.
Regulation is mediated by the lactose repressor protein which has
a
(1)
lactose binding site
(2)
DNA binding site (specific for operator sequence)
g.
When lactose is absent, repressor binds to DNA at the lac operator
and prevents RNA polymerase from binding to the lac promoter.
Transcription is blocked.
h.
When lactose is present, repressor binds to it and is then unable to
bind to DNA. RNA polymerase can now bind to lac promoter and
transcription begins.