Download Transcription and Translation

Transcription and Translation
Genes
The structure of a prokaryote gene
Gene
• Consensus sequences
• - 10 and -25 upstream from the start
• 3’ TAC – start or initiator codon
• Establishes the reading frame – ORF –
open reading frame
• Bases grouped in 3’s triplet code
• Proceeds to stop ( ATT , ATC, ACT or
termination sequence in prokaryotes
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
Promoters are at the beginning of the Gene
• RNA polymerase recognizes a binding site in front of the
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5’
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
TTGACA3’ ( Watson strand) which means on the reading
strand 3’ AACTGT5’ ( Crick strand)
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 - Continued
• Genes can also specify t- RNA or r- RNAs
• The gene begins with a start triplet and ends with a
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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
Prokaryote Genes are
• Continuous
• 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
Transcription Begins
• 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
RNA POLYMERASE
RNA Polymerase
• There are approximately 2000
molecules of RNA Polymerase per
bacterial cell
• E. coli RNA polymerase is composed of
six subunits with a molecular weight of
over 400,000 Da
RNA POLYMERASE
• Enzyme has been described as a claw
• The Beta portions of the claw form the
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pincers
The Alpha portion are on the other end of the
claw
The Omega portion is wrapped around the
Beta portion
RNA Polymerase Action
• Does not require a primer in comparison
to DNA Polymerase
• Binds to the promoter sequence and
opens the double strands of DNA
• Builds a chain of RNA by connecting the 5’
end of a nucleotide to the 3’ end of the
nucleotide in front of it
Language
• RNA polymerase utilizes the sequence of
the template strand in DNA language to
build a complementary copy of RNA
• The strand that is the template is referred
to as the transcribed strand
• The strand with the same sequence as
RNA with the exception of U instead of T is
referred to as the coding strand
RNA Polymerase Structure – Core
enzyme
Polymerase sub units
• α2: the two α subunits assemble the enzyme and
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recognize regulatory factors.
These compose the core enzyme
β: this has the polymerase activity (catalyzes the
synthesis of RNA) which includes chain initiation
and elongation.
β': binds to DNA (nonspecifically).
ω: restores denatured RNA polymerase to its
functional form in vitro. It has been observed to offer
a protective/chaperone function to the β' subunit
RNA Polymerase Holoenzyme
Sigma Factors
• A sigma factor (σ factor) are proteins that are
required for the binding of the RNA polymerase to
the promoter so that transcription can be initiated.
They are also active in the elongation process.
Transcription and the Initiation
Complex
• The core enzyme binds to the holoenzyme
• This is called promoter recognition
• The sigma factors first bins to the -10
region
• When the Beta pincer closes around the
DNAto form the active site channel
around the DNA, this opens the strands
Rifampin
• The initiation somplex can be blocked
by the antibiotic Rifampin
• Rifampin binds to the RNA Polymerase
at the active site channel is such a way
that it blocks the elongation of the RNA
Mutations, Antibiotics, and
Antibiotic Resistance
Initiation, Elongation, Termination
Transcriptional overview
• http://vcell.ndsu.nodak.edu/~christjo/vcell
/animationSite/transcription/elongation.ht
m
Rho independent( Factor
dependent)
• The termination site consists of two sequences
• The first is an inverted repeat
• When the inverted repeat is transcribed it forms
a hairpin
• The inverted repeat is followed by a string of
AAAAAAAAAAAs
• The RNA molecule will have terminal UUUUs
• AU pairs are less stable and this causes the
release of the RNA molecule from the DNA
Termination- Factor Independent
Factor Dependent
• Rho Dependent ( Rho the most universal factor
in prokaryote cells). There are other factors
• Rho functions in the synthesis of RNAs that are
not being translated
• Rho forms a ring that binds to a sequence in the
RNA called the rut site
• Binds to the RNA and affects the action of RNA
polymerase
Eukaryote Transcription and
Transcription Factors
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 sequences that are removed after
transcription.
• The acceptor stem
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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’
t-RNA
t- RNA
• 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
t- RNA
Common Features
• 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.
tRNA
• tRNAs attach to a
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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
Ribosomal genes
• 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
Ribosomal RNA
• Combines with specific proteins to form
ribosomes
• Serves as a site for protein synthesis
• Associated enzymes and factors control
the process of translation
Prokaryote Ribosomes
• 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.
30s and 50s Ribosomal
subunits
Prokaryote ribosomal RNA
Type of
rRNA
Approxim
ate
number
of
nucleotid
es
Subunit
Location
16s
1,542
30s
5s
120
50s
23s
2,904
50s
Prokaryote ribosomes – polysomesthe 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
Translation
• 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
T RNA and Amino acyl t RNA
synthetase( Preparation of tRNAs)
• The enzyme amino acyl t RNA Synthetase
connects the amino acid to its specific t
RNA to form cognate t RNA
• Hydrolyzes ATP to transfer the amino acid
to the 3’OH end of the t RNA
• The acceptor arm of the t RNA ends in the
sequence CCA
The Ribosome
• There are four significant positions on
the ribosome
• EPAT
• 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
Ribosomal Sites
• T site – the 5’ end of the messenger RNA
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enters the ribosome
A site – acceptor site -the relationship of the
mRNA to the ribosome is stabilized at this
site – the amino acylated t RNA is bound to
the mRNA
P site the peptide bonds are formed between
the amino acids on the t RNAs
E site - The mRNA moves to the final position
on the ribosome as the tRNA is released
EPAT
Ribosomal structure
Translation Initiation Region
• Should contain the initiator codon( start)
AUG, may also be GUG
• This may be at the start of the gene or in an
Untranslated leader sequence upstream of
Gene ( UTR)
• Shine Delgarno named for two scientists who
discovered it at -35 binds to the 30s ribosomal
subuit ( 16srRNA)
Archaea Initiation
• Combination of Eukarya and Bacteria
Initiation
• Archaea and Eukarya have more initiation
factors than Bacteria
• Archaea uses formylated methionine
Initiation
Elongation
Elongation
Translocase
• Moves the tRNA from the A site to the P
site
• This displaces the t RNA on the P site to
the E site
• Elongation factors function at this point
• Energy required from the hydrolysis of
GTP
Peptidyl transferase
• Forms the peptide bonds between the
amino acids attached to the t RNAs
• Forms the nascent polypeptide chain
Termination
• http://www.phschool.com/science/biology
_place/biocoach/translation/term.html
• Release factors function in termination
( RF1 and 3)
• GTP is required for energy
• The termination or stop codon enters the
A site( UAA, UAG, UGA)
• Recognized by the ribosome
Termination
From Gene to polypeptide
Codon chart
•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
WOBBLE
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
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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?
Hypermutation
• One possible explanation is hypermutation
• A starving bacterium has the ability to generate
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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
Spontaneous mutations
Types
1. 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
2. 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.
3. Insertions or deletions cause frame shifts – the
code shifts over the number of bases inserted or
deleted
Mutation Types
• Errors in replication due
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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
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Depurination
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
Frame Shifts
• 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
Mutagenesis
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Any agent that directly
damages DNA, alters its
chemistry, or interferes
with repair mechanisms
will induce mutations
Base analogs
Specific mispairing
Intercalating agents
Ionizing radiation
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
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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
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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
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Requires a sensitive system
Mutations are rare
One in about every 107 – 1011
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
Ames Test
• 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
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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
Recombination repair
• The DNA repair for which there is no remaining template is
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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