Download RNA

F
From
Gene
G
to
t Protein
P t i
z
Beadle and Tatum
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Analyzed Fungi Neurospora crassa mutants
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Mutants were UNABLE to grow without Arginine
(an amino acid)
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Other biochemical experiments indicated:
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Precursor Æ Ornithine Æ Citrulline Æ Arginine
Each biochemical reaction requiring an enzyme
Hypothesis:
ONE gene ONE enzyme
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Beadle and Tatum
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Mutants could be classified into one of three groups
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z
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Grew with ornithine supplements
Grew with citrulline supplements
Grew only with arginine supplements
Indicates that a gene is required for each step
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Precursor Æ Ornithine Æ Citrulline Æ Arginine
g
Modifications to Beadle and Tatum’s
Hypothesis
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Reasons for modifications:
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z
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Enzymes can contain multiple protein and/or RNA subunits
Not all proteins are enzymes
ONE gene ONE polypeptide hypothesis
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Still not entirely accurate as we will learn
z Genes can encode for RNAs that are NOT used as a code
for protein (ie NOT mRNA)
z A single gene can be used to generate multiple different
proteins
DNA Æ RNA Æ Protein
P t i
z
DNA Æ RNA
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Transcription
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The p
production of ribonucleic acid using
g DNA as a
template
RNA Æ Protein
P t i
z
Translation
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The production of a polypeptide using an RNA as a
template
Transcription and Translation
occur in BOTH p
procaryotic
y
and
eucaryotic cells
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Procaryotic Cells
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z
No Nucleus
z Transcription and Translation are
coupled
Eucaryotic Cells
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Nucleus
z Transcription and Translation
occur in different cellular
locations
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One strand of DNA is used as a
Template to produce a single
strand of RNA
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RNA is produced in the 5’ to 3’
direction
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The template DNA strand is read
in the 3’ to 5’ direction
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In protein production, the template
RNA (termed a messenger RNA)
is read in the 5’ to 3’ direction
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3 nucleotides, or a codon, code for
a single
i l amino
i acid
id
Genetic Code
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z
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4 RNA nucleotides
20 amino acids
Theoretical
z 2 letter code
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3 letter code
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4 x 4 = 16 possibilities NOT ENOUGH
4 X 4 X 4 = 64 possibilities
ibiliti ENOUGH
Experimental
z Nirenberg produced an artificial poly U RNA and performed
translation in a test tube
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Produced a polypeptide with just phenylalanine
G
Genetic
ti Code
C d
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Start
z AUG
Stop
z UAA, UAG, UGA
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Non overlapping
z 9 nucleotides contains only 3 codons!
z UGC AGU CCA
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Redundant
z Some amino acids are coded for by multiple codons
z
Proline
ƒ
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CCU, CCC, CCA, CCG
The g
genetic code is essentially
y the same in all 3 domains:
bacteria, archaea, eucaryotes
T
Transcription
i ti
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The process by
Th
b which
hi h a DNA template
l
iis used
d to b
build
ild a strand
d
of RNA
z
RNA Polymerase
z The enzyme responsible for the condensation/ dehydration
reactions that build an RNA
z
Occurs in the 5’ to 3’ direction
z
Occurs in three stages
g
z Initiation
z Elongation
z Termination
Transcription
p
z
Initiation
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A region off the double stranded DNA serves to
attract the RNA polymerase. This region of DNA
is termed the Promoter Region
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Promoter regions frequently contain a TATA box
Transcription Factors:
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z
Proteins that interact with the nucleotides of the DNA
Recruit the RNA polymerase
Transcription
p
z
Initiation (cont’d)
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The transcription initiation complex results from
the transcription factors and the RNA
polymerase
z
The double helix is temporarily
p
y unwound as the
RNA polymerase produces an RNA strand in the
5’ to 3’ direction
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The DNA template is read by the RNA
polymerase in the 3’ to 5’ direction
T
Transcription
i ti
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Elongation
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Addition of nucleotides to the 3’ end of the growing RNA
continues
Termination
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z
Procaryotes
z Triggered by a sequence in the DNA called the terminator
E
Eucaryotes
t
z Not entirely understood, but the RNA is cleaved from the
RNA polymerase following transcription of a poly
adenylation signal AAUAAA
RNA processing
i
z
Only occurs in eucaryotic cells
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Alteration of the mRNA ends
RNA splicing
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Pre-mRNA
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RNA Processing
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mRNA
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5’ end
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A modified guanine nucleotide is added
3’ end
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After the polyadenylation site AAUAAA a string
of adenine nucleotides is added (between 50 –
250)
mRNA structure
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5’ cap
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5’ UTR
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Untranslated region
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Runs from the transcription start site (TSS) to the start codon (AUG)
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Coding region
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3’ UTR
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Stop codon until the poly (A) tail
Poly(A) tail
Alt
Alternative
ti RNA splicing
li i
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Observation: The actual size of many genomic regions used to
produce an RNA transcript are MUCH larger than the actual
mRNA used in the production of protein
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IIntrons
t
z Non-coding segments of an RNA that are removed prior to
translation
z “Intervening”
Intervening
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Exons
z The coding portion of an RNA that is used for translation
Intron
RNA splicing
li i
Exon
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The removal of introns and the splicing together of exons
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The reaction is catalyzed by a multi-protein RNA complex termed
the Spliceosome
Pre-mRNA
mRNA
Translation
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mRNA
z
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Ribosome
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z
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Transcribed and processed
RNA from a gene
Proteins
Ribosomal RNA
tRNA
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Transfer RNA
ƒ
ƒ
Contains an anticodon
Contains an amino acid
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The Single stranded RNA that is the transfer RNA forms hydrogen
bonds amongst its nucleotides giving it a 3 dimensional shape
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Amino acid forms a covalent bond with the 3’ end of the tRNA
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The anticodon, 3’
3 to 5
5’ forms hydrogen bonds with the codon, 5’
5 to 3
3’
of the mRNA
Addition of the amino acid to the tRNA is catalyzed by an
enzyme: aminoacyl-tRNA synthetase
z
20 different aminoacyltRNA synthetases
z
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One for each amino acid
The addition of the amino
acid to the tRNA uses
ATP
T
Translation
l ti
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Initiation
z Ribosome, mRNA, tRNA
association
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Elongation
z Covalent peptide bond
formations between successive
amino acids
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Termination
e
at o
z Dissociation of ribosome,
mRNA, and tRNA
Translation Initiation
z
Small subunit binds to the mRNA
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The start codon is recognized by the small subunit
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The initiator tRNA containing the amino acid Met is recruited
z
The large
g subunit binds in a p
process that utilizes GTP,, forming
g the
translation initiation complex
T
Translation
l ti
Elongation
El
ti
T
Translation
l ti
Termination
T
i ti
z
A stop codon, UAA, UAG, UGA recruits a protein release factor
z
The bond between the tRNA and the polypeptide is hydrolyzed by the
release factor
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Dissociation of the mRNA, ribosome, and release factor
Polyribosomes or Polysomes
z
More often than not, in both eucaryotic and procaryotic cells
and single mRNA contains many ribosomes simultaneously
producing
g polypeptide
y
Procaryotic
P
ti cells
ll can couple
l
transcription and translation
DNA Æ RNA Æ protein
t i
z
Transcription
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RNA processing
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Translation
Cytoplasmic and ER bound
ribosomes
ib
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Rib
Ribosomes
start iin the
h cytoplasm
l
z
A signal
i
l sequence iin th
the N tterminus
i
off th
the protein,
t i ttermed
d th
the
signal peptide will target a protein for the ER to become part of
the Endomembrane System (as discussed earlier in the
course))
z
The Signal-Recognition Particle, a multi-protein RNA complex
facilitates binding of the ribosome to the ER and entry of the
synthesizing protein into the ER where it can then proceed to
the Golgi apparatus via a transport vesicle
Types of RNAs
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Messenger RNA
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Transfer RNA
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RNAs that are p
part of the Signal
g
Recognition
g
Particle
snoRNA
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RNAs that are part of the Spliceosome
SRP RNA
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Enzymatic RNAs that make up a portion of the Ribosome
Small nuclear RNA (snRNA)
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Functions in translation by bringing amino acids to the mRNA using an
anticodon
Ribosomal RNA
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Codes for polypeptide
Process ribosomal RNAs
siRNA, miRNA
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Involved in Gene Regulation
P i t Mutation
Point
M t ti
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Single changes
in the DNA
sequence
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Can h
C
have
drastic effects
Types
yp of Point mutations
z
Base-Pair substitution
z A change
g in composition
p
or nucleotide type
yp at a single
g location
z
z
z
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Silent: The substitution codes for the SAME amino acid
Missense Mutation: the substitution codes for another amino acid
Nonsense Mutation: the substitution codes for a STOP codon
causing premature termination of the polypepetide
INDELs or Frameshift mutations
z The insertion or deletion of one or more base pairs
p
z
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NO Frameshift: if 3 base pairs (or some multiple of 3) is added,
then the reading frame will be the same
Frameshift: a change in all subsequent codons
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THE CAT ATE THE DOG
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THE CAT CAT ATE THE DOG
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THE CAT CAT ETH EDO G
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Frameshift can cause
z
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MISSENSE
NONSENSE