Organization of bacterial chromosome Prokaryotic DNA replicate

Organization of bacterial chromosome Prokaryotic DNA replicate, transcription & translation

by Angelia Teo (Jan 10) 1

Bacterial chromosome, structure & organization

Prokaryotic DNA replication, transcription, translation

Prokaryotic regulation of gene expression

Mutations and Selection

Extra-chromosomal elements.

- Bacteriophages

- Plasmid DNA

2by Angelia Teo (Jan 10)

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.

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.

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.

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


Single circular chromosome

haploid

naked DNA

▪ no histone proteins

~4 million base pairs

▪ ~4300 genes

▪ 1/1000 DNA in eukaryote


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


Molecules of double-stranded DNA Usually circular Tend to be shorter Contains a few thousand unique genes Mostly structural genes Single origin of replication


The bacterial chromosome is found in region called the nucleoid (not membrane-bounded-so the DNA is in direct contact with the cytoplasm)


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.


Bacterial Genome is haploid, single chromosome

Not all bacteria have a single circular chromosome: some bacteria have multiple circular chromosomes, and many bacteria have linear chromosomes and linear plasmids.

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


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.


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.


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

DNA supercoiling Topoisomerases twist the DNA and control

degree of supercoiling


The length of a typical bacterial operon (usually about 3

genes), is about as long as the entire bacterial cell !


http://www.wadsworth.org/databank/ecoli.htm


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.


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.

DNA molecules that replicate as discrete genetic units in bacteria are called replicons.

Extrachromosomal replicons: - bacteriophages- plasmids (non-essential replicons)

These determine resistance to antimicrobial agents or production of virulence factors.



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.

http://en.wikipedia.org/wiki/File:Phage.png


A plasmid is an extra-chromosomal DNAmolecule separate from the chromosomal DNAwhich 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.

http://en.wikipedia.org/wiki/File:Plasmid_(english).svg

http://en.wikipedia.org/wiki/DNA

http://en.wikipedia.org/wiki/Chromosome

http://en.wikipedia.org/wiki/Bacterium

http://en.wikipedia.org/wiki/Eukaryote

They are haploid (no masking). Only 1 set of

genes

New generation is produced every 20 mins

Easy to grow in ENORMOUS NUMBERS

Individual members of these large populations are

GENETICALLY IDENTICAL

Advantages


Genetic material of bacteria is DNA.

Bacterial viruses (bacteriophages) have DNA

or RNA as genetic material.

DNA must replicate accurately so that progeny inherit all of

the specific genetic determinants (genotype) of

the parental organism.

Specific DNA expression under a particular set of

growth conditions determines the observable characteristics (phenotype) of the organism.


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.


DNA is a double-stranded helix; two strands are anti-parallel.

Double helix is stabilized by H bonds between purine& pyrimidine bases on the opposite strands. A pairs Tby 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 intra-molecular base-pairing.


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.

.

X

Y

Autoradiograph of intact

replicating chromosome

of E coli. Bacteria were

radioactively labeled with

tritiated thymidine


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.


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!


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.


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.


Transcription & TranslationProkaryotic 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.

Prokaryotic cell Eukaryotic cell 43by Angelia Teo (Jan 10)

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.


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).


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.


The Genetic codeThe "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.

• 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.


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.


Charlebois, R. 1999. Organization of the Prokaryotic Genome. ASM Press, Washington, D.C. Casjens, S. 1998. The diverse and dynamic structure of bacterial genomes. Ann. Rev. Genet. 32:

339-377. Casjens, S. 1999. Evolution of the linear DNA replicons of the Borrelia spirochetes. Curr. Opin.

Microbiol. 2: 529-534. Chen, C. 1996. http://www.ym.edu.tw/ig/cwc/end_troubles/End_Troubles.html Jumas-Bilak et al. 1998. Unconventional genomic organization in the alpha subgroup of the

Proteobacteria. J. Bacteriol. 180: 2749-2755. Kobryn K, Chaconas G. 2001. The circle is broken: telomere resolution in linear replicons. Curr

Opin Microbiol. 4(5): 558-564. Suwanto, A., and S. Kaplan. 1989. Physical and genetic mapping of the Rhodobacter sphaeroides

2.4.1 genome: presence of two unique circular chromosomes. J. Bacteriol. 171: 5850-5859. Suwanto, A and S. Kaplan. 1992. Chromosome transfer in Rhodobacter sphaeroides: Hfr

formation and genetic evidence for two unique circular chromosomes. J. Bacteriol. 174: 1135-1145.

Trucksis et al. 1998. The Vibrio cholerae genome contains two unique circular chromosomes. Proc. Natl. Acad. Sci. USA 95: 14464-14469.

Volff, J.-N., and J. Altenbuchner. 2000. A new beginning with new ends: linearisation of circular chromosomes during bacterial evolution. FEMS Microbiol. Lett. 186: 143-150.

Yang CC, Huang CH, Li CY, Tsay YG, Lee SC, Chen CW. 2002. The terminal proteins of linear Streptomyces chromosomes and plasmids: a novel class of replication priming proteins. Mol Microbiol. 43(2): 297-305.


Organization of bacterial chromosome Prokaryotic DNA replicate

Documents

Transcript of Organization of bacterial chromosome Prokaryotic DNA replicate