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Eukaryotic RNA polymerase (RNP) exists in three unique forms,
each of which transcribes different types of genes.
Each enzyme is larger and more complex than the prokaryotic RNP.
RNP II is responsible for the production of mRNA.
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Central dogma of genetics.
Genetic information is encoded as a linear sequence of deoxyribonucleotides on one of the two strands of DNA (the template strand).
Transcription produces a "messenger" RNA (mRNA) complementary to the template.
Translation occurs on ribosomes,
where the message on mRNA determines the sequence of amino acids that are assembled
into proteins.
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Electron micrograph visualizing the process of transcription.
A gene being transcribed shows RNA molecules growing progressively longer from top to
bottom.
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Khorana (Nobel 1968) developed a technique to
synthesize long RNA molecules consisting of short sequences (di-, tri-, and tetranucleotides) repeated many times.
These repeating copolymers yield a predictable combination of potential codons.
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Some exceptions to the genetic code
are found in mitochondrial DNA (mtDNA) of yeast and humans, as well as in the DNA of
some single-celled organisms.
Some of the changes, such as the UGA codon in row 1.
involve only a shift in recognition of the third, or wobble, position.
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rII (rapid lysis)
locus of phage T4
causes a shift in the reading frame
of all subsequent downstream codons, and potential incorrect amino acids.
The expressed protein is usually nonfunctional,
and a T4 phage with such a frameshift mutation cannot reproduce on E. coli K12.
three insertions occur in tandem, the reading frame is restored after the
insertion.
Such a mutation produces a protein with enough functionality to restore the phage's ability to infect K12.
genetic code
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The nearly universal genetic code serves as a dictionary for translation from mRNA to amino acid.
The code is degenerate: many amino acids specified by more than one codon; only tryptophan and methionine are encoded by a single codon.
In addition to codons that specify amino acid,
there is one “start” (or “initiator”) codon
(AUG, which also encodes methionine) and three “stop” codons.
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Electron micrograph of a hybrid molecule (heteroduplex) formed by hybridization
between the template DNA strand of a gene and its mature mRNA transcript in the chicken ovalbumin
gene.
Seven DNA introns, A–G, produce unpaired loops.
The heteroduplex loops are formed because they contain introns
which cannot pair with the mRNA.
heteropolymer.html: 13_04-heteropolymer.jpg
Calculations of the frequency of possible codons produced using a heteropolymer
composed of a ratio of 1A:5C.
There is a 1/6 possibility for an A and a 5/6 chance for a C to occupy each position in the triplet.
By examining the percentages of amino acids incorporated into the protein synthesized, probable base composition for some codons can be proposed.
Proline appears 69% of the time, so it may ne encoded by CCC (57.9%) and one codon consisting of 2C:1A (11.6%).
Histidine, at 14%, is probably coded by one 2C:1A (11.6%) and one 1C:2A (2.3%).
Threonine, at 12%, is likely coded by only one 2C:1A.
Asparagine and glutamine each seem to be coded by one of the 1C:2A codons, and lysine by AAA.
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splicing.
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As a first step in deciphering the genetic code, short artificial mRNA
homopolymer sequences of
UUUUUU ..., AAAAAA ..., or CCCCCC
were used as the template to synthesize polypeptides using radioactively labeled amino acids.
Poly U was found to incorporate 14C-phenylalanine, indicating that the codon
for phenylalanine is UUU.
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The base composition of RNA produced after phage infections resembles that of the phage DNA
and not that of the bacterial host.
This suggests that RNA synthesis may be a intermediate step in protein synthesis.
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Most eukaryotic genes contain introns.
The ovalbumin gene of chickens is mostly "silent", containing seven introns
that together are twice as long as the exon segments.
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Most genes contain introns. In extreme cases such as the dystrophin gene,
less than 1 percent of the gene sequence is retained in the mRNA.
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An mRNA sequence initiated at two different AUG positions out of frame with one another will give rise to
overlapping genes
that specify two distinct polypeptides.
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The relative positions of the sequences encoding seven polypeptides in the phage φχ174 (phi chi 174):
Three overlapping genes (A, C, and D) serve to specify seven different polypeptides.
The genome of this virus is small: the circular DNA consists of 5386 nucleotides,
which should encode a maximum of 1795 amino acids.
polynucleotide_phosphorylase.html: 13_03-polynucleotide_phosphorylase.jpg
Nirenberg and Matthaei (Nobel 1968) produced RNA sequences from high concentrations of
ribonucleoside diphosphates.
The sequence of nucleotides produced can be controlled by varying the concentration of rNDPs.
This RNA served as the "messenger" to synthesize polypeptide chains in vitro.
The system also included the enzyme polynucleotide phosphorylase, ribosomes, tRNAs, and amino acids.
repeating_copolymers.html: 13_T03-repeating_copolymers.jpg
The repeating copolymers yield predictable triplet codons.
These synthetic mRNAs can be used to incorporate amino acids into proteins in vitro.
These data, combined with those drawn from composition
assignment
studies
and triplet binding,
specific assignments of the codons can be completed.
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Group III introns contain a GU dinucleotide "donor" sequence at the 5' end of the intron,
and an AG "acceptor" sequence at the 3' end.
A set of small nuclear RNAs (snRNAs designated U1, U2, ... U6) bind to the donor sequence, forming a complex called a spliceosome.
continue
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As with group I splicing, two transesterification reactions excise the intron,
which in this case forma a loop structure called a lariat.
The exons are then ligated to form the mature mRNA.
animation
splicing.html: 13_13a-splicing.jpg
Pre-rRNAs contain group I introns and are called ribozymes:
they can catalyze their own splicing by self-excision in a series of 2 transesterification
reactions.
Folding of the RNA chain exposes a Guanosine nucleotide in an active site within the intron.
next
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In reaction 1, the 3'-OH group of Guanosine binds to the nucleotide adjacent to the 5' end of the
intron.
next
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An exchange of OH groups exposes a new 3'-OH on the left-hand exon and a phosphate on the right exon,
leading to reaction 2.
next
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A 2nd exchange of OH groups results in excision of the intron and ligation (joining) of
the two exon regions.
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Multiple strands of RNA are transcribed along a DNA template in E. coli.
Ribosomes attach to the nascent mRNA and initiate translation simultaneously.
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After initiation, the σ (sigma) subunit dissociates from the holoenzyme,
and chain elongation proceeds under the direction of the core enzyme,
until it eventually encounters a termination sequence.
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RNA polymerase catalyzes the insertion of
ribonucleoside triphosphate molecules in the 5' to 3' direction, linked together by phosphodiester bonds,
forming an antiparallel DNA/RNA duplex.
No primer is required in this initiation process.
Next:
elongation.
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Multiple strands of RNA are transcribed along a DNA template in the
newt
Notophthalmus viridescens.
No ribosomes are seen, since translation occurs in the cytoplasm in eukaryotes,
after RNA processing.
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In E. coli, the σ (sigma) subunit of RNA polymerase binds to the promoter
region on the DNA.
Next:
initiation.
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About 50 of the 64 codons were assigned using the triplet binding
assay.
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Triplet binding assay.
Ribosomes can bind to three-ribonucleotide sequences (codon) in vitro,
which in turn bind to a complementary anticodon within tRNA carrying a specific,
radioactively charged amino acid.
The whole complex can be bound to nitrocellulose filter, and assayed for the charged amino
acids.
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In many cases, the first two letters of the genetic code
are more critical in specifying an amino acid.
For example, the codon for valine (val) only depends on the first 2 letters (GU).
The 3rd position of the codon can "wobble": a single tRNA can pair with more than one codon in mRNA.
U at the 1st position (5') of the tRNA anticodon may pair with A or G at the 3rd position (3') of the mRNA codon, and G may likewise pair with U or C.
Inosine (I), a modified base found in tRNA, may pair with C, U, or A.