Transcript MB206_fhs_lnt_001.1_AT_May09

Organization of bacterial chromosome Prokaryotic
DNA replicate, transcription & translation
by Angelia Teo (May 09)
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 Bacterial chromosome, structure & organization
 Prokaryotic DNA replication, transcription, translation
 Prokaryotic regulation of gene expression
 Mutations and Selection
 Extra-chromosomal elements.
- Bacteriophages
- Plasmid DNA
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the genome of prokaryotes is not in a separate
compartment, haploid. Single chromosome: it is located
in the cytoplasm (although sometimes confined to a
particular region called a “nucleoid”). Prokaryotes
contain no membrane-bound organelles; their only
membrane is the membrane that separates the cell form
the outside world. Nearly all prokaryotes are unicellular.
Eukaryotes are defined as having their genetic material enclosed
in a membrane-bound nucleus, separate from the cytoplasm. In
addition, eukaryotes have other membrane-bound organelles
such as mitochondria, lysosomes, and endoplasmic reticulum.
almost all multicellular organisms are eukaryotes.
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Prokaryotes are haploid, and they contain a
single circular chromosome. In addition,
prokaryotes often contain small circular DNA
molecules called “plasmids”, that confer useful
properties such as drug resistance. Only circular
DNA molecules in prokaryotes can replicate.
Eukaryotes are often diploid, and eukaryotes have linear
chromosomes, usually more than 1.
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In prokaryotes, translation is coupled to
transcription: translation of the new RNA
molecule starts before transcription is
finished.
In eukaryotes, transcription of genes in RNA occurs in the
nucleus, and translation of that RNA into protein occurs
in the cytoplasm. The two processes are separated from
each other.
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Bacteria review
 one-celled organisms
 prokaryotes
 reproduce by mitosis
▪ binary fission
 rapid growth
▪ generation every ~20 minutes
▪ 108 (100 million) colony overnight!
 dominant form of life on Earth
 incredibly diverse
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Single circular chromosome
 haploid
 naked DNA
▪ no histone proteins
 ~4 million base pairs
▪ ~4300 genes
▪ 1/1000 DNA in eukaryote
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No nuclear membrane
 chromosome in cytoplasm
 transcription & translation are coupled
together
▪ no processing of mRNA
 no introns
 but Central Dogma
still applies
▪ use same
genetic code
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Molecules of double-stranded DNA
Usually circular
Tend to be shorter
Contains a few thousand unique genes
Mostly structural genes
Single origin of replication
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The bacterial chromosome is found in region
called the nucleoid (not membrane-boundedso the DNA is in direct contact with the
cytoplasm)
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Bacterial Genome is haploid, single chromosome
The circularity of the bacterial chromosome was elegantly
demonstrated by electron microscopy in both Gram
negative bacteria (such as Escherichia coli) and Gram
positive bacteria (such as Bacillus subtilis).
 Bacterial plasmids were also shown to be circular.
 Linear chromosomes found in Gram-positive
Borrelia & Streptomyces.
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Not all bacteria have a single circular chromosome: some bacteria have
multiple circular chromosomes, and many bacteria have linear
chromosomes and linear plasmids.
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Bacterial chromosomal DNA is usually a circular
molecule that is a few million nucleotides in
length
 Escherichia coli  4.6 million base pairs
 Haemophilus influenzae  1.8 million base pairs
A typical bacterial chromosome contains a few
thousand different genes
 Structural gene sequences (encoding proteins)
account for the majority of bacterial DNA
 The nontranscribed DNA between adjacent genes are
termed intergenic regions
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Chromosomal Map of Bacteria
Circular genetic map of E coli.
Positions of representative
genes are indicated on inner
circle. Distances between genes
are calibrated in minutes, based
on times required for transfer
during conjugation. Position of
threonine (thr) locus is arbitrarily
designated as 0 minutes, and
other assignments are relative
to thr.
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The Complete
Sequence of
Escherichia coli
Chromosome
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Most, but not all, bacterial species contain circular
chromosomal DNA.
A typical chromosome is a few million base pairs in
length.
Most bacterial species contain a single type of
chromosome, but it may be present in multiple copies.
Several thousand different genes are interspersed
throughout the chromosome.
One origin of replication is required to initiate DNA
replication.
Short repetitive sequences may be interspersed
throughout the chromosome.
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Typical bacterial chromosome must be
compacted about 1,000-fold
Bacterial DNA is not wound around histone
proteins to form nucleosomes
Proteins important in forming loop domains
 Compacts DNA about 10-fold
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DNA supercoiling
 Topoisomerases twist the DNA and control
degree of supercoiling
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The length of a typical bacterial operon (usually about 3
genes), is about as long as the entire bacterial cell !
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The operon model of prokaryotic gene regulation was proposed by Fancois Jacob and
Jacques Monod. The lac operon is an operon required for the transport and
metabolism of lactose in Escherichia coli and some other enteric bacteria. It consists
of three adjacent structural genes, a promoter, a terminator, and an operator. The lac
operon is regulated by several factors including the availability of glucose and of
lactose. The regulator does not have to be adjacent to other genes in the operon. If the
repressor protein is removed, transcription may occur.
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Operons are either
inducible or repressible
according to the control
mechanism. Seventy-five
different operons
controlling 250 structural
genes have been
identified for E. coli. Both
repression and induction
are examples of negative
control since the
repressor proteins turn
off transcription.
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DNA molecules that replicate as discrete
genetic units in bacteria are called replicons.
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Extrachromosomal replicons:
- bacteriophages
- plasmids (non-essential replicons)
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These determine resistance to antimicrobial
agents or production of
virulence factors.
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Bacteriophage (from
'bacteria' and Greek
φάγειν phagein "to
eat")
is any one of a number of
viruses that infect
bacteria. The term is
commonly used in its
shortened form, phage.
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A plasmid is an extra-chromosomal DNA
molecule separate from the chromosomal DNA
which is capable of replicating independently of
the chromosomal DNA. In many cases, it is
circular and double-stranded. Plasmids usually
occur naturally in bacteria, but are sometimes
found in eukaryotic organisms.
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They are haploid (no
masking). Only 1 set of
genes
New generation is
produced every 20 mins
Advantages
Easy to grow in
ENORMOUS NUMBERS
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Individual members of
these large populations are
GENETICALLY IDENTICAL
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Genetic material of bacteria is
DNA.
DNA must replicate accurately
so that progeny inherit all of
the specific genetic
determinants (genotype) of
the parental organism.
by Angelia Teo (May 09)
Bacterial viruses
(bacteriophages) have DNA
or RNA as genetic material.
Specific DNA expression
under a particular set of
growth conditions determines
the observable characteristics
(phenotype) of the organism.
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Nucleic acids are polynucleotides, consist of repeating nucleotide units
Each nucleotide contains one phosphate group, one sugar (pentose
or deoxypentose) and one base (purine or pyrimidine).
Phosphodiester bonds link the 3'-OH of one nucleotide sugar to the
5'-OH group of the adjacent nucleotide sugar.
In DNA the sugar is D-2-deoxyribose; in RNA the sugar is D-ribose.
RNA has a hydroxyl group on the 2' carbon of the sugar.
In DNA the purine bases are adenine (A) and
guanine (G), and the pyrimidine bases are thymine
(T) and cytosine (C).
In RNA, uracil (U) replaces thymine.
Chemically modified purine and pyrimidine bases
are found in some bacteria and bacteriophages.
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 DNA is a double-stranded helix; two strands are antiparallel.
 Double helix is stabilized by H bonds between purine
& pyrimidine bases on the opposite strands. A pairs T
by 2 H bonds; G pairs C by 3 H bonds.
 Two strands in DNA helix are complementary, ie.
dsDNA contains equimolar amounts of purines (A +
G) and pyrimidines (T + C), with A = T and G = C.
The mole fraction of G + C in DNA varies widely
among different bacteria.
 DNA is supercoiled and tightly packaged.
 The extent of sequence homology between DNAs
from different microorganisms determines how
closely related they are (eg. 16sRNA sequence)
 RNA exists as a single-stranded molecule; forms
hairpin loops (secondary structure) due to intramolecular base-pairing.
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DNA Replication in Bacteria
 The DNA replicates semiconservatively:
- Each strand in dsDNA serves as a
template for synthesis of a new
complementary strand.
- Result: daughter dsDNA molecule contains one old polynucleotide strand
and one newly synthesized strand.
 Replication of chromosomal DNA in
bacteria starts at a specific chromosomal
site called the origin of replication and
proceeds bi-directionally until the process is
completed.
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Autoradiograph of intact
replicating chromosome
of E coli. Bacteria were
radioactively labeled with
tritiated thymidine
X
Y
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DNA Replication in Bacteria
 DNA replication is initiated whenever cells divide, so in rapidly
growing bacteria a new round of chromosomal replication begins
before an earlier round is completed.
 The origin regions specifically and transiently associate with
the cell membrane after initiation of DNA replication. Membrane
attachment directs separation of daughter chromosomes.
 Time required for replication of the entire chromosome is
about 40 minutes (500 – 1000 nucleotides / sec)
 Replicated chromosomes are partitioned into each of the
daughter cells.
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Central Dogma of Molecular Biology
How does the sequence of
a strand of DNA correspond
to the amino acid sequence
of a protein?
• DNA codes for RNA production.
• RNA codes for protein production.
• Protein does not code protein, RNA
or DNA production.
The end.
Or in the words of Francis Crick:
Once information has passed into
protein, it cannot get out again!
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Revision of the "Central Dogma"
 CAN
go back from RNA to DNA (reverse transcriptase)
 RNA can also make copies of itself (RNA polymerase)
 Still NOT possible from Proteins back to RNA or DNA
 Not known mechanisms for proteins making copies of themselves.
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Gene Expression
 Expression of genetic determinants in bacteria involves the
unidirectional flow of information from DNA to RNA to
protein.
 Two processes involved are transcription and translation.
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Transcription & Translation
Prokaryotic vs Eukaryotic cells
In a prokaryotic cell, which does not contain a nucleus, this
process happens at the same time.
In Eukaryotic cells, occur at different
cell compartments.
by Angelia Teo
(May 09)
Prokaryotic
cell
Eukaryotic cell
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Transcription
 The DNA-directed synthesis of RNA is called transcription.
 Transcription produces RNA molecules that are complimentary
copies of one strand of DNA.
 Only one of the dsDNA strands can serve as template for
synthesis of a specific mRNA molecule.
 mRNAs transmit information from DNA, and each mRNA in
bacteria function as a template for synthesis of one or more
specific proteins.
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Translation
 The process by which the nucleotide sequence of an mRNA molecule
determines the primary amino acid sequence of a protein.
 Ribosomes are complexes of ribosomal RNAs (rRNAs) and several
ribosomal proteins.
 Ribosomes with the aid of transfer RNAs (tRNAs), amino-acyl tRNA
synthesases, initiation factors and elongation factors are all involved in
translation of each mRNA into corresponding polypeptide (protein).
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Translation
 Initiated at an AUG codon for methionine.
 Codons are translated sequentially in mRNA from 5' to 3'.
 The corresponding polypeptide chain / protein is assembled
from the amino terminus to carboxy terminus.
 The sequence of amino acids in the polypeptide is, therefore,
co-linear with the sequence of nucleotides in the mRNA and the
corresponding gene.
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The Genetic code
The "universal" genetic code employed by most organisms is a triplet code and it
determines how the nucleotides in mRNA specify the amino acids in the
polypeptide.
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61 of 64 possible trinucleotides
(codons) encode specific amino
acids.
3 remaining codons (UAG, UAA or
UGA) code for termination of
translation (nonsense codons = do
not specify any amino acids)
Exceptions:
1) UGA as a tryptophan codon in some
species of Mycoplasma and in
mitochondrial DNA.
2) Few codon differences in
mitochondrial DNAs from yeasts,
Drosophila, and mammals.
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Gene expression occurs in 2 steps:
Transcription of the information encoded in DNA into a molecule of RNA
Translation of the information encoded in mRNA into a defined sequence of
amino acids in a protein.
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The sequence of one strand of DNA is
5’ GGGTAAGCTTATCCCGTA 3’
3’ CCCATTCGAATAGGGCAT 5’
The sequence of the complementary strand from 5’ to 3’
is
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A) CCCATTCGAATAGGGCAT
B) TACGGGATAAGCTTACCC
C) GGGTAAGCTTATCCCGTA
D) ATGCCCTATTCGAATGGG
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The following is the sense strand of the DNA sequence. Give the amino acid
sequence of the protein generated
after translation.
3’ TACCCCATGATGGTAGGGTTAGTAGGGTTATCCATGGGG 5’
5’ ATGGGGTACTACCATCCCAATCATCCCAATAGGTACCCC 3’
TRANSCRIPTION
5’ AUGGGGUACUACCAUCCCAAUCAUCCCAAUAGGUACCCC 3’
TRANSLATION
Met Gly Tyr Tyr His Pro Asn His Pro Asn Arg Tyr Pro
5’AUG GGG UAC UAC CAU CCC AAU CAU CCC AAU AGG UAC CCC 3’
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Charlebois, R. 1999. Organization of the Prokaryotic Genome. ASM Press, Washington, D.C.
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Jumas-Bilak et al. 1998. Unconventional genomic organization in the alpha subgroup of the
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