Introduction to Microbial Genetics Microbiology 221Document Transcript
Introduction to Microbial Genetics Microbiology 221
The Race for the Double Helix
Rosalind Franklin and Maurice Wilkins at Kings College
Studied the A and B forms of DNA
Rosalind’s famous x-ray crystallography picture of the B form held the secret, but she didn’t realize its significance
The Race for the Double Helix
Watson and Crick formed an unlikely partnership
A 22 year old PhD and a 34 year old “want to be” PhD
embarked on a model making venture at Cambridge
Used the research of other scientists to determine the nature of the double helix
Nucleic Acid Composition DNA and RNA
DNA – Basic Molecules
Purines – adenine and guanine
Pyrmidines – cytosine and thymine
Sugar – Deoxyribose
Phosphate phosphate group
http://www. dnai .org/index. htm - DNA background
Two polynucleotide strands joined by phosphodiester bonds( backbone)
Complementary base pairing in the center of the molecule
A= T and C G – base pairing. Two hydrogen bonds between A and T and three hydrogen bonds between C and G.
A purine is bonded to a complementary pyrimidine
Bases are attached to the 1’ C in the sugar
At opposite ends of the strand – one strand has the 3’hydroxyl, the other the 5’ hydroxyl of the sugar molecule
DNA Structure http:// www.johnkyrk.com/DNAanatomy.html - DNA structure
Double helix ( continued)
The double helix is right handed – the chains turn counter-clockwise.
As the strand turn around each other they form a major and minor groove.
The is a distance of .34nm between each base
The distance between two major grooves is 2.4nm or 10 bases
The diameter of the strand is 2nm
Complementary Base Pairing
Adenine pairs with Thymine
Cytosine pairs with Guanine
The end view of DNA
This view shows the double helix and the outer backbone with the bases in the center.
An AT base pair is highlighted in white
Double helix and anti-parallel
DNA is a directional molecule
The complementary strands run in opposite directions
One strand runs 3’-5’
The other strand runs 5’ to 3’
( the end of the 5’ has the phosphates attached, while the 3’ end has a hydroxyl exposed)
Polynucleotide – nucleic acid - Single stranded molecule that can coil back on itself and produce complementary base-pairing ( t- RNA)
Four bases in RNA are Adenine and Guanine ( purines) and Cytosine and Uracil( pyrimidines)
Sugar – ribose
Three types of RNA
nc- non coding RNA’s
Coiling maintained by molecules similar to the coiling in eukaryotes
Circular ds molecule
Some Special Cases
Borrelia burgdoferi ( Lyme Disease )has a linear chromosome
Other bacteria have multiple chromosomes
Agrobacterium tumefaciens ( Produces Crown Gall disease in plants) has both circular and linear
E. coli – most often studied in molecular biology of prokaryotes
The genes of E. coli are located on a circular chromosome of 4.6 million basepairs. This 1.6 mm long molecule is compressed into a highly organized structure which fits inside the 1-2 micrometer cell in a format which can still be read by the gene expression machinery.
Bacterial DNA and Supercoiling
Bacterial DNA is supercoiled by DNA gyrase. Chemical inhibition of gyrase without allowing the cells to reprogram gene expression relaxes supercoiling and expands the nucleoid, suggesting that supercoiling is one of the tools used to compress the genome
Coiling maintained by Gyrase
Relaxation of the coils by Topoisomerase
DNA is more highly organized in eukaryote cells
The DNA is associated with proteins called histones.( eukaryotes)
These are small basic proteins rich in the amino acids lysine and/or arginine
There are five histones in eukaryote cells, H1, H2A, H2B,H3 and H4.
The nature of DNA replication was elucidated by Meselson and Stahl
Meselson and Stahl experiment
Grew bacteria in heavy Nitrogen – N-15
Transferred bacteria to N-14
Before bacteria reproduce in new media, all bacteria contain heavy DNA
Samples were taken after one round of replication and two round of replication
Each original strand serves a template or pattern for the replication of the new strand.
The new strand contains one original and a newly synthesized strand
Multiple linear chromosomes
Each chromosome has more than one origin of replication
Approximately 1400 x as long as bacterial DNA
Multiple replicons on a chromosome
Oris along the length – every 10 to 100 um
Replication forks and bubbles are formed. Replication proceeds bidirectionally until the bubbles meet
This shortens the length of time necessary to replicate eukaryote chromosomes
The process of elongation occurs at a speed of 50-100 base pairs/minute as compared to 750 to 1000 base pairs/ minute
The origin of replication and replication forks
During the S phase, there are 100 replication complexes and each one contains as many as 300 replication forks. These replication complexes are stationary. The DNA threads through these complexes as single strands and emerges as double strands.
Fourteen DNA polymerases have been observed in human beings as compared to three in E. coli.
There is an origin of replication
Two replication forks are formed
Replication occurs around the circle until they have opened and copied the entire chromosome
Replicon- contains an origin and is replicated as a unit
Ori – Origin of replication
Characteristics used to define Origins:
The position on the DNA at which replication start points (see right) are found.
A DNA sequence that when added to a non-replicating DNA causes it to replicate.
A DNA sequence whose mutation abolishes replication.
A DNA sequence that in vitro is the binding target for enzyme
When the double helix of DNA, which is composed of two strands, separates, helicase makes these two strands rotate around each other.
The DnaB protein is the helicase most involved in replication, but the n’ protin may also participate in unwinding.
The single stranded binding proteins SSBP help to keep the strand open
But there is a problem due to the topological reason that the unreplicated part ahead of the replication fork will rotate around its helical axis when the two strands separate at the replication fork
It causes strong strain in the helix (1). Thus, it is impossible to unlink the double helical structure of DNA without disrupting the continuity of the strands.
In order to perform unraveling of a "compensating winding up" DNA, enzymes are required (1). Topoisomerase changes the linking number as well as catalyzes the interconversionn of other kinds of topological isomers of DNA (2).
Initiation a. oriC - origin of chromosomal replication Recognized by DnaA protein - only recognizes if GATC sites are fully methylated Binding of DnaA allows DnaB to open complex b. DnaB is the replication helicase c. Strand separation by helicase d. SSB (single-stranded binding) protein keeps strands apart e. DNA gyrase - a topoisomerase - puts swivel in DNA which allows strands to rotate and relieve strain of unwinding
Recall that DNA double helix is tightly wound structure and that bases lie between the two backbones. If these bases are the template for new strand, how do the appropriate enzymes reach these bases? By the unwinding of the helix.
An enzyme called helicase catalyzes the unwinding of short DNA segments just ahead of the replication fork. The reaction is driven by the hydrolysis of ATP.
As soon as duplex is unwound, SSB (single-stranded binding protein) binds to each of the separated strands to prevent them from base-pairing again. Therefore, the bases are exposed to the replication system.
The unwinding of the duplex would cause the entire DNA molecule to swivel except for the action of a topoisomerase (DNA gyrase) which introduce breaks in the DNA just ahead of the unwinding duplex. These breaks are then rejoined after a few revolutions of the duplex.
The need for a primer
When DNA template is exposed, DNA synthesis must begin. But DNA polymerases not only need a template but also a primer for replication to proceed. Where does the primer come from?
After observations that RNA synthesis is required for DNA synthesis, it was discovered that the synthesis of DNA fragments requires a short length of RNA as a primer. Primosome (complex of 20 polypeptides) makes RNA primers in E. coli
Formation of the Primer
Primosome contains primase
Primosome moves along DNA duplex in 3'>5' direction (with respect to lagging strand; follows replication fork) even though primer is made in 5'>3' direction (Note: The symbol ">" indicates the direction; that is, the primer is made from 5' to 3'.) n' protein removes SSB in front of primosome
DnaB protein organizes some components of primosome and prepares DNA for primase Primase forms the primer
DNA POLYMERASE III
Complex that synthesizes most of the DNA copy contains the DNA polymerase enzyme and other proteins
The gamma delta complex and the B subunits of the holoenzyme bind it to the template and the primer
The alpha subunit carries out the actual polymerization reaction
All of the proteins form a huge complex called the replisome
DNA polymerase III
This is a stationary complex that probably attached to the plasma membrane.
The DNA moves through the replisome and is copied
Elongation of the chain
dCTP dCMP +
Energy is supplied for biosynthesis by the cleaving of the phosphate bond
Elongation proceeds in 5' > 3' direction and requires 1) all 4 deoxyribonucleoside 5'-triphosphates (dATP, dGTP, dCTP, dTTP), 2) Mg+ ions, 3) a primer made of nucleic acid, and 4) a DNA template.
Rate of elongation = 750 - 1000 nucleotides per second Rate of formation of initiation complex = 1-2 minutes
Elongation DNA polymerase I, II and III in E .col i DNA polymerase III holoenzyme - complex of 7 polypeptides
Replisome - primosome and 2 DNA polymerase III - synthesizes DNA on both strands simultaneously without dissociating from DNA
DNA polymerase III catalyzes the addition of deoxyribonucleotide units to end of the DNA strand with release of inorganic pyrophosphate (PPi) (DNA)n residues + dNTP <> (DNA)n + 1 residues + PPi Attachment of new units is by their a-phosphate groups to a free 3'-hydroxyl end of preexisting DNA chain.
The lagging strand and discontinuous replication
The replication on the 5’ to 3’ strand differs
The template strand still must be read from 3’ to 5’
The reading begins at the replication fork
Occurs at the same time as the synthesis of the lagging strand
Same steps in synthesis of DNA
But DNA is synthesized in pieces about 1000 to 2000 bases in length. These are known as Okazaki fragments
After the lagging strand has been duplicated by the formation of Okazaki fragments, DNA Polymerase I or RNase H removes the RNA primer. Polymerase I synthesizes the complementary DNA to fill the gap resulting from the RNA delection.
The polymerase removes one nucleotide at a time and then replaces it
AMP( RNA nucleotide) replaced by dAMP( DNA nucleotide)
Ligase can catalyze the formation of a phosphodiester bond given an unattached but adjacent 3'OH and 5'phosphate.
This can fill in the unattached gap left when the RNA primer is removed and filled in.
The DNA polymerase can organize the bond on the 5' end of the primer, but ligase is needed to make the bond on the 3' end.
The End of Replication
DNA replication stops when the polymerase complex reaches a termination site on the DNA in E. coli
The Tus protein binds to the ter site and halts replication.
In many prokaryotes the replication process stops when the replication forks meet
ColE1 is a naturally occurring plasmid of E. coli . Its replication is controlled independently of the replication of the host chromosome.
Two plasmids with the same origin of replication can not coexist in the same cell.
The ColE1 origin, defined by molecular genetic methods, is in a region from which two RNAs are transcribed.
An active RNase H gene is required for ColE1 replication. RNase H cleaves the RNA II transcript. The remaining RNA serves as primer for initiation of replication.
RNA I binds to 5' sequences of RNA II via pseudoknots and regular complementary pairing. This binding is stabilized by the ROP or ROM protein.
The binding prevents changes in the conformation of RNA II that would otherwise result in RNAse H cleavage.
Rolling Circle Replication – Occurs in Conjugation in E. coli.
How can one account for the high fidelity of replication?
The answer is based on the fact that DNA Polymerase absolutely requires 3'-OH end of base-paired primer strand on which to add new nucleotides.
DNA polymerase III has 3' > 5' exonuclease activity. It was discovered that DNA polymerase III actually proofreads the newly synthesized strand before continuing with replication. When incorrect nucleotide is incorporated, DNA polymerase III, by means of the 3' > 5' exonuclease activity, "backs up" and hydrolyzes off the incorrect nucleotide. The correct nucleotide is then added to the chain and elongation is resumed.
All 3 DNA polymerases have 3'>5' exonuclease activity
Proofreading ability - 1 error in 10 million
Exonucleases and repair
DNA polymerase I also has 5'>3' exonuclease activity which removes RNA primer and 5'>3' polymerase activity which fills in the gap
This causes a single-stranded break in the DNA - called a nick DNA ligase repairs nick by creating a phosphodiester bond
Genes and Gene Expression
Genes are written in a code consisting of groups of three letters called triplets.
There are four letters in the DNA alphabet. There are 64 possible arrangements of the four letters in groups of three
The triplets specify amino acids for the synthesis of proteins from the information contained in the gene
Genes can also specify t- RNA or r- RNAs
The gene begins with a start triplet and ends with a stop. The bases between the start and the stop are called an open reading frame, ORF.
The information in the gene is transcribed by RNA polymerase.
It reads the gene from 3’ to 5’
The template strand is now referred to as the CRICK strand and the nontemplate strand is now known as the WATSON strand
DNA sequences are stored in data bases as the WATSON strand
Reference - COLD SPRING HARBOR - 2003
Promoters are at the beginning of the Gene
RNA polymerase recognizes a binding site in front of the gene. This is referred to as upstream of the gene.
The direction of transcription is referred to as downstream
Different genes have different promoters. IN E. coli the promoters have two functions
The RNA recognition site for transcription which is the consensus sequence for prokaryotes is
5’ TTGACA3’ ( Watson strand) which means on the reading strand 3’ AACTGT5’ ( Crick strand)
The Pribnow Box and Shane -Dalgarno
The RNA binding site has a consensus sequence of
5’ TATAAT 3’ ( -) and 3’ ATATTA 5’ (+)
This is where the DNA begins to become unwound for transcription
The initially transcribed sequence of the gene may not reflect doing but may be a leader sequence.
The prokaryotes usually contain a consensus sequence known as the Shane Delgarno which is complememtary to the 16s rRNA on the ribosome
( small subunit )
The leader sequence also may regulate transcription
The structure of a prokaryote gene
Prokaryote Genes are
They do not contain introns like eukaryote genes
The gene consists of codons that will determine the sequence of amino acids in the protein
At the end of the gene there is a terminator sequence rather than an actual stop
The terminator may be at the end of a trailer sequence located downstream from the actual coding region of the gene
The Gene begins with
DNA is read 3’ to 5’ and m RNA is synthesized 5’ to 3’
3’ TAC is the start triplet
This produces a complementary mRNA message 5’ AUG 3’ –
Groups of three bases in the messenger RNA formed are referred to as CODONS
There is wobble in the DNA code – This is a protection from mutations
More than one codon can specify the same amino acid
Note arginine - CGU, CGC,CGA, CGG all code for arginine – only the third base in the codon changes
There are two additional codons for arginine as well AGA and AGG these reflect the degenerate nature of the code
Genes for t RNAs and r RNAs
The genes for t RNAs have a promoter and transcribed leader and trailer sequence that are removed prior to their utilization in translation. Genes coding for tRNA may code for more than a single tRNA molecule
The segments coding for r RNAs are separated by spacer sequencs that are removed after transcription.
The acceptor stem includes the 5' and 3' ends of the tRNA.
The 5' end is generated by RNase P
The 3' end is the site which is charged with amino acids for translation.
Aminoacyl tRNA synthetases interact with both the acceptor 3' end and the anticodon when charging tRNAs.
The anticodon matches the codon on mRNA and is read
3’ to 5’
Found in the cytoplasm
Amino acyl t- RNA synthetase is an enzyme that enables the amino acid to attach to t-RNA
Also activates the t- RNA
Clover leaf has a stem for attachment to the amino acid and an anticodon on the bottom of the clover leaf
a CCA trinucleotide at the 3' end, unpaired
four base-paired stems, and
One loop containing a T-pseudoU-C sequence and another containing dihydroU.
tRNAs attach to a specific amino acid and carry it to the ribosome
There are 20 amino acids
61 different codons for these amino acids and 61 tRNAs
The anticodon is complementary to the codon
Binds to the codon with hydrogen bonds
Very similar to the structure of protein genes
tRNA and rRNA genes
The genes for rRNA are also similar to the organization of genes coding for proteins
All rRNA genes are transcribed as a large precursor molecule that is edited by ribonucleases after transcription to yield the final r RNA products
Combines with specific proteins to form ribosomes
Serves as a site for protein synthesis
Associated enzymes and factors control the process of translation
Ribosomes are small, but complex structures, roughly 20 to 30 nm in diameter, consisting of two unequally sized subunits, referred to as large and small which fit closely together as seen below.
A subunit is composed of a complex between RNA molecules and proteins; each subunit contains at least one ribosomal RNA (rRNA) subunit and a large quantity of ribosomal proteins.
The subunits together contain up to 82 specific proteins assembled in a precise sequence.
Prokaryote ribosomal RNA 50s 2,904 23s 50s 120 5s 30s 1,542 16s Subunit Location Approximate number of nucleotides Type of rRNA
Prokaryote ribosomes – polysomes- the process of translation
Prokaryote transcription and translation
Prokaryote transcription and translation take place in the cytoplasm
All necessary enzymes and molecules are present for the transcription and translation to take place
A molecule of messenger RNA binds to the 30S ribosome
( small ribosomal unit) at the Shine Dalgarno sequence
This insures the correct orientation for the molecule
The large ribosomal sub unit locks on top
There are four significant positions on the ribosome
When the 5’ AUG 3’ of the mRNA is on the P site the t-RNA with the anticodon, 5’UAG3’ forms a temporary bond to begin translation
From Gene to polypeptide
E. Coli Gene Map
Mutations in DNA
May be characterized by their genotypic or phenotypic change
Mutations can alter the phenotype of a microorganisms in different ways
Mutations can involve a change in the cellular or colonial morphology
Types of Mutations
Conditional mutations are those mutations that are expressed only under specific environmental conditions ( temperature)
Biochemical mutations are those that can cause a change in the biochemistry of the cell
( these may inactivate a biochemical pathway)
These mutants are referred to as auxotrophs because they cannot grow on minimal media
Prototrophs are usually wild type strains capable of growing on minimal media
Two types of mutations
Spontaneous mutations – These occur without a causative agent during replication
Induced mutations are the result of a substance referred to as a mutagen
Cairns reports that a mutant E. coli strain unable to use lactose is able to regain its ability to use the sugar again – should this be referred to as adaptive mutation?
One possible explanation is hypermutation
A starving bacterium has the ability to generate multiple mutations with special mutator genes that enable them to form bacteria with the ability to metabolize lactose
This is an interesting theory still under investigation
A purine substitutes for a purine or a pyrimidine substitutes of a pyrimidine. This type of mutation is referred ta as a transition. Most of these can be repaired by proofreading mechanisms
A pyrimidine substituted for by a purine is referred to as a transversion. These are rarer due to steric problems in the DNA molecule such as pairing purines with purines.
Insertions or deletions cause frame shifts – the code shifts over the number of bases inserted or deleted
Erors in replication due to base tautomerization
AT and CG pairs are formed when keto groups participate in hydrogen bonds
In contrast enol tautomers produce AC and GT base pairing
Spontaneous mutations – another cause
A purine nucleotide can lose its base
It will not base pair normally
It will probably lead to a transition type mutation after the next round of replication.
Cytosine can be deaminated to uracil which can then create a problem
Additions and deletions change the reading frame.
The hypothetical origin of deletions and insertions may occur during replication
If the new strand slips an insertion or addition may occur
If the parental slips a deletion may occur
Any agent that directly damages DNA, alters its chemistry, or interferes with repair mechanisms will induce mutations
Base analogs are structurally similar to normal nitrogenous bases and can be incorporated into the growing polynucleotide chain during replication.
The expression of mutations
Forward mutations – a mutation from the wild type to a mutant form is called a forward mutation
Reversion-If the organism regains its wild type characteristics through a second mutation
Back mutation – The actual nucleotide sequence is converted back to the original
Suppressor mutation – overcomes the effects of the first mutation
More on mutations
Point mutations – caused by the change in one DNA base
Silent mutations – mutations can occur which cause no effect – this is due to the degeneracy of the code ( more than one base coding for the same amino acid)
Missense mutation – changes a codon for one amino acid into a codon for another amino acid
Nonsense – In eukaryotes the substitution of a stop into the sequence of a normal gene
Detection and isolation of mutants
Requires a sensitive system
Mutations are rare
One in about every 10 7 – 10 11
Replica plating is a technique that is used to detect auxotrophs
It distinguishes between wild type and mutants because of their ability to grow in the absence of a particular biosynthetic end product
Replica plating allows plating on minimal media and enriched media from the same master plate
The selection of auxotorph revertants
The lysine auxotrophs ( Lys-) are treated with a mutagen such as nitroquanidine or uv light to produce revertants
Developed by Bruce Ames
Used to test for carcinogens
A mutational reversion assay based upon mutants of Salmonella typhimurium
DNA repair mechanisms
Type I -Excision repair
Corrects damage which causes distortions in the double helix
A repair endonuclease or uvr ABC endonuclease removes the damaged bases along with some bases on either side of thee lesion
The usual gap is about 12 nucleotides long. It is filled by DNA polymerase and ligase joins the fragments.
This can remove Thymine-Thymine dimers
A special type of repair utilizes glycosylases to remove damaged or unnatural bases yielding the results discussed above
Mutations and repair
Type II – Removal of lesion
Thymine dimers and alkylated bases are often repaired directly
Photoreactivation is the repair of thymine dimers by splitting them apart into separate thymines with the aid of visible light in a photochemical reaction catalyzed by the enzyme photolyase
Light repair - phr gene - codes for deoxyribodipyrimidine photolyase that, with cofactor folic acid, binds in dark to T dimer. When light shines on cell, folic acid absorbs the light and uses the energy to break bond of T dimer; photolyase then falls off DNA
Dark repair of mutations
Dark repair Three types 1) UV Damage Repair (also called NER - nucleotide excision repair) Excinuclease (an endonuclease; also called correndonuclease [correction endo.]) that can detect T dimer, nicks DNA strand on 5' end of dimer (composed of subunits coded by uvrA , uvrB and uvrC genes). UvrA protein and ATP bind to DNA at the distortion. UvrB binds to the UvrA-DNA complex and increases specificity of UvrA-ATP complex for irradiated DNA. UvrC nicks DNA 8 bases upstream and 4 or 5 bases downstream of dimer. UvrD (DNA helicase II; same as DnaB used during replication initiation) separates strands to release 12-bp segment. DNA polymerase I now fills in gap in 5'>3' direction and ligase seals.
The Effects of uv light
Post replication repair
If T dimer not repaired, DNA Pol III can't make complementary strand during replication. Postdimer initiation - skips over lesion and leaves large gap (800 bases). Gap may be repaired by enzymes in recombination system - lesion remains but get intact double helix.
Successful post replication depends upon the ability to recognize the old and newly replicated DNA strands
This is possible because the newly replicated DNA strand lack methyl groups on their bases, whereas the older DNA has methyl groups on the bases of both strands.
The DNA repair system cuts out the mismatch from the non- methylated strand
The DNA repair for which there is no remaining template is restored
RecA protein cuts a piece of template DNA from a sister molecule and puts it into the gap or uses it to replace a damaged strand
Rec A also participates in a type of inducible repair known as SOS repair.
If the DNA damage is so great that synthesis stops completely leaving many gaps, the Rec A will bind to the gaps and initiate strand exchange.
It takes on a proteolytic funtion that destroys the lexA repressor protein which regulates genes involved in DNA repair and synthesis