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Transcript of 2013 10 31-From DNA to Protein
8/15/2019 2013 10 31-From DNA to Protein
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Chapter 6
From DNA to Protein:How Cell Read the Genome
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Genetic information directs the synthesis of protein
The central dogma of molecular biology
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Genes can be expressed with different efficienciesUntranscribed portions
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Nucleotide
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The chemical structure of RNA differs slightly
from that of DNA
3’
5’
phosphodiester
bond
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Uracil forms a base pair with adenine
2 hydrogen bonds
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RNA molecules can form intramolecular base pairs
and fold into specific structures
Base-pair with complementary sequences
Conventional
base-pair interactions
Nonconventional
base-pair
interactions
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Non-Coding strand Anti-sense strand
Template strand
Transcription produces an RNA complementary
to one strand of DNA
Coding strand
Sense strand
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Sense & antisense strand
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DNA duplication by DNA polymerase
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Transcription by RNA polymerase
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DNA is transcribed by the enzyme RNA polymerase
ATP
CTP
UTPGTP
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Transcription can be visualized in the electron microscope
Gene 1 Gene 2
rRNAs
RNA polymeraseDNA
Ribosomal proteins
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RNA polymerase vs DNA polymerase
RNA polymerase DNA polymerase
Ribonucleotides Deoxyribonucleotides
Without primer With primer
1/104 error rate 1/107 error rate
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Types of RNA produced in cells
messenger RNA
ribosomal RNA
microRNA
transfer RNA
n o n - m e s s e
n g e r R N A
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Signals in the sequence of a gene tell bacteria
RNA polymerase where to start and stop transcription
Bacterial RNA polymerase
Chain elongation
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Bacterial RNA polymerase
Bacterial promoters and terminators
have specific nucleotide sequences
that are recognized by RNA polymerase
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The direction of transcription is determined by the orientation of the promoter at the beginning of each gene
Some genes are transcribed using
one strand DNA as a template, whereas others are
transcribed using the other DNA strand
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The three RNA polymerases in eucaryotic cells
mRNA
sRNAs
sRNAs
T b i i i i RNA l II
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To begin transcription, eucaryotic RNA polymerase II
requires a set of general transcriptional factors
-25
TATA-binding protein
Dramatic local distortion in the DNA
Dephosphorylated form
Transcription initiation complex
Phosphorylate the tail
of RNA polymerase II
Allow the template strandto be exposed by
ATP hydrolysis
TATA bi di t i (TBP) bi d t TATA b
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TATA-binding protein (TBP) binds to TATA box sequences
and distorts the DNA
TATA box DNA
TBP (TATA-binding protein)
B f th b t l t d RNA l l d
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Before they can be translated, mRNA molecules made
in the nucleus move out into the cytoplasm via pore
in the nuclear envelope
Pores in
nuclear envelope
TEM
Ph h l ti f RNA l II ll
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(1) Capping
(2) Polyadenylation
(3) Splicing
Phosphorylation of RNA polymerase II allows
RNA-processing proteins to assemble on its tail
TFIIH
Eucaryotic mRNA molecules are modified by
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Eucaryotic mRNA molecules are modified by
capping and polyadenylation (1)
Start after 25 nucleotides
has been polymeized
(1) To increase the stability of the eucaryotic mRNA molecule
(2) To aid its export from the nucleus to the cytoplasm
(3) To identify the RNA molecule as an mRNAFunctions:
Eucaryotic mRNA molecules are modified by
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Eucaryotic mRNA molecules are modified by
capping and polyadenylation (2)
1
2 34
5
CH3
Eucaryotic and bacterial genes are organized differently
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Eucaryotic and bacterial genes are organized differently
Promoter
Intron is longer than exon
Most human genes are broken into exons and introns
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Most human genes are broken into exons and introns
3 exons
26 exons
Special nucleotide sequences signal
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Special nucleotide sequences signal
the beginning and the end of an intron
RNA splicing might occurred before or after polyadenylation
R: A or G
Y: C or U
N: A or C or G or U
long short
Branch point of the lariat
intron-exon
boundary (border)
RNA splicing
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RNA splicing
An introns forms a branched structure during splicing
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An introns forms a branched structure during splicing
5’
3’ 5’
Branch point of the lariat
Spliceosome
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Spliceosome
Small nuclear RNAs (snRNAs) + Proteins
= Small nuclear ribonucleoprotein particles
(snRNPs)
The -tropomyosin gene can be spliced in different ways
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The tropomyosin gene can be spliced in different ways
(1) Many different protein to be produced from the same gene by alternative splicing(2) 60% of human genes probably undergo alternative splicing
A specialized set of RNA-binding proteins signal that
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A specialized set of RNA binding proteins signal that
a mature mRNA is ready for export to the cytoplasm
Recognizes and exports
only completed mRNAs
Life times depends on
(1) Nucleotide sequence (3’ untranslated sequence)
(2) The type of cell
Exon junction complex (EJC)
Mark completed RNA splices
3’ untranslated region
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3 untranslated region
Procaryote and eucaryotes handle
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y y
their RNA transcripts differently
Transcription in procaryotic or eucaryotic cells (1)
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p p y y ( )
Transcription in procaryotic or eucaryotic cells (2)
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p p y y ( )
Bacteria Eucaryotic cells
Single type Three types (I, II, III)
X General transcription factors
Short Long
RNA polymerase
Accessory proteins
Nontranscribed DNA between genes
Small RNAs
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(1) siRNA (small interfering RNA)
(2) miRNA (microRNA)(3) piRNA (piwi-interacting RNA)
miRNA
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siRNA
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The nucleotide sequence of an mRNA is translated into
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the amino acid sequence of a protein
via the genetic code
Stop
codonsStart
codon
T
h r e e - n u c l e o t i d e
c o d o n s
A m i n o
a c i d s
UUU codes for phenylalanine
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Heinrich MatthaeiMarshall Nirenberg
Messages of mixed repeating sequences further
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narrowed the coding possibilities
Gobind Khorana
The Nobel Prize in Physiology or Medicine 1968
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Interpretation of the genetic code and its function in protein synthesis
An RNA molecule can be translated
i th ibl di f
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in three possible reading frames
Reading frame
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Frame shift
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tRNA molecules are molecular adaptors,
linking amino acids to codons
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linking amino acids to codons
pseudouridine
dihydrouridine
L-shape molecule
Wobble base-pairing
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61 codons
31 anticodons 20 amino acids
The genetic code is translated by means of two adaptors
that act one after another
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that act one after another
Aminoacyl-tRNA synthase
Charged tRNA
Charging
12 Anticodon
Ribosomes are found in the cytoplasm of a eucaryotic cell
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Attached to the ER
TEM
Free in thecytosol
Ribosome
A ribosome is a large complex of four RNAs and
more than 80 proteins
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more than 80 proteins
(rRNA)
(rRNA)
Catalyzes the formation
of polypeptide chain
Matches the tRNA to
the codon of the mRNA
1/3
2/3
Each ribosome has a binding site for mRNA
and three binding sites for tRNA
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and three binding sites for tRNA
Large subunit Small subunit
Large subunit
Small subunit
Exit
peptidyl-tRNA
aminoacyl-tRNA
Translation takes place in a four-step cycle
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Catalyzed by an enzymatic site
in the large subunit
Large subunit
Ribosomal RNAs give the ribosome its overall shape
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Protein
Large subunit of a bacterial ribosome
Catalytic site for peptide bond formation
Rate of sedimentation in an ultracentrifuge
Initiation of protein synthesis in eucaryotes requires
initiation factors and a special initiator tRNA
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initiation factors and a special initiator tRNA
Start codon
E P A
E P A
E P A
or formylmethionine in bacteria
only this charged RNA
can binds to the P-site
In the final phase of protein synthesis, the binding of
release factor to an A-site bearing a stop codon
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release factor to an A site bearing a stop codon
terminates translation
Stop codons
UAG
UGA
UAA
A single procaryotic mRNA molecule can encode
several different proteins
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several different proteins
Operons
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Proteins are translated by polyribosomes
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Polyribosomes (Polysomes)
Inhibitors of procaryotic protein synthesis
are used as antibiotics
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The proteosome degrades short-lived and
misfolded proteins
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Active site of
the proteases
Ubiquination for protein degradation
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Ubiquitin
Protein production in a eucaryotic cell
requires many steps
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Many proteins require additional modification
to become fully functional
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Glycosylation (> 100 kinds)
An RNA world may have existed
before modern cells arose
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An RNA molecule can in principle guide
the formation of an exact copy of itself
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Ribozyme
Ribozyme – RNA molecules that possess catalytic activity
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Ribozyme – RNA molecules that possess catalytic activity
A ribozyme is an RNA molecule that possesses
catalytic activity
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Biochemical reactions that can be catalyzed by ribozymes
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Could an RNA molecule catalyze its own synthesis?
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RNA may have preceded DNA and proteins in evolution
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