Download Lecture 7: Life`s Information Molecule II

BIO 2, Lecture 7
LIFE’S INFORMATION
MOLECULE II: TRANSCRIPTION
Overview:
The Flow of Genetic Information
• The information content of DNA is in the
form of specific sequences of nucleotides
• The DNA inherited by an organism leads to
specific traits by dictating the synthesis of
proteins
• Gene expression, the process by which DNA
directs protein synthesis, includes two
stages: transcription and translation
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
• Changes in the nucleotide sequence of
DNA can lead to changes in the amino
acid sequence of proteins
• The genotype of an organism is comprised
of the genes that it carries
• The phenotype of an organism is comprised
of its physical and behavioral traits
• An organism’s phenotype is dictated, to a
large extent, by its genotype
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
Genotype: 47(+21) XY
Phenotype: Down Syndrome
Genes do not control every
aspect of phenotype ...
• In 1909, British physician Archibald
Garrod first suggested that genes
dictate phenotypes through enzymes that
catalyze specific chemical reactions
• He thought symptoms of an inherited
disease reflect an inability to synthesize
a certain enzyme
• Linking genes to enzymes required
understanding that cells synthesize and
degrade molecules in a series of steps, a
metabolic pathway
• George Beadle and Edward Tatum
exposed bread mold to X-rays, creating
mutants that were unable to survive on
minimal medium as a result of inability to
synthesize certain molecules
• Using crosses, they identified three
classes of arginine-deficient mutants,
each lacking a different enzyme
necessary for synthesizing arginine
• They developed a one gene–one enzyme
hypothesis, which states that each gene
dictates production of a specific enzyme
EXPERIMENT
Growth:
Wild-type
cells growing
and dividing
No growth:
Mutant cells
cannot grow
and divide
Minimal medium
RESULTS
Classes of Neurospora crassa
Wild type
Condition
Minimal
medium
(MM)
(control)
MM +
ornithine
MM +
citrulline
MM +
arginine
(control)
Class I mutants Class II mutants Class III mutants
CONCLUSION
Wild type
Gene A
Gene B
Gene C
Class I mutants Class II mutants Class III mutants
(mutation in
(mutation in
(mutation in
gene A)
gene B)
gene C)
Precursor
Precursor
Precursor
Precursor
Enzyme A
Enzyme A
Enzyme A
Enzyme A
Ornithine
Ornithine
Ornithine
Ornithine
Enzyme B
Enzyme B
Enzyme B
Enzyme B
Citrulline
Citrulline
Citrulline
Citrulline
Enzyme C
Enzyme C
Enzyme C
Enzyme C
Arginine
Arginine
Arginine
Arginine
• Some proteins aren’t enzymes, so
researchers later revised the hypothesis:
one gene–one protein
• But proteins with quarternary structure
are encoded by more than one gene, so
Beadle and Tatum’s hypothesis was again
revised as the one gene–one polypeptide
hypothesis
• But now we know that each gene can
encode more than one polypeptide due
to a phenomenon called alternative
splicing ...
• So now we have the one gene–one or
more polypeptides hypothesis
• RNA is the intermediate between genes
and the proteins for which they code
• Transcription is the copying of one strand
of the double-stranded DNA into a singlestranded RNA molecule
• Transcription produces messenger RNA
(mRNA)
• Translation is the synthesis of a
polypeptide from the mRNA on a ribosome
RNA
polymerase
Chromosomes are like a
library of books (in the
form of DNA molecules)
that cannot be checked out
But an mRNA copy of
some of the pages of
some of the books
(genes) are made in a
process called
transcription
Ribosomes
Ribosomes then read
the instructions in
the RNA molecules
to build proteins in a
process called
translation
• In prokaryotes, transcription and
translation take place in the same space
(the cytosol) and the mRNA produced by
transcription is immediately translated
without more processing
• In a eukaryotic cell, the nuclear envelope
separates transcription from translation
• Eukaryotic RNA transcripts are modified
through RNA processing to yield finished
mRNA
The central dogma is the concept that cells
are governed by a cellular chain of command:
DNA RNA protein
TRANSCRIPTION
TRANSLATION
DNA
mRNA
Ribosome
Polypeptide
(a) Bacterial cell
Nuclear
envelope
DNA
TRANSCRIPTION
Pre-mRNA
RNA PROCESSING
mRNA
TRANSLATION
Ribosome
Polypeptide
(b) Eukaryotic cell
• How are the instructions for assembling
amino acids into proteins encoded by the
DNA?
• There are 20 amino acids, but there are
only four nucleotide bases in DNA
• How many bases correspond to an amino
acid?
• The flow of information from gene to
protein is based on a triplet code: a
series of non-overlapping, threenucleotide “words” called codons
• Example: The triplet 5’-AGT-3’ in a gene
results in the placement of the amino acid
serine in the polypeptide coded by the
gene
• Another example: 5’-GGG-3’ codes for the
animo acid glycine
• During transcription, one of the two DNA
strands is copied into mRNA
• During translation, the codons in the
mRNA are read in the 5 to 3 direction
• Each codon specifies the amino acid to be
placed at the corresponding position along
a polypeptide
DNA
molecule
Gene 2
Gene 1
Gene 3
DNA
template
strand
TRANSCRIPTION
mRNA
Codon
TRANSLATION
Protein
Amino acid
DNA
coding
strand
• All 64 codons were deciphered by the
mid-1960s (43 = 64)
• Of the 64 triplets, 61 code for amino
acids; 3 triplets are “stop” signals to end
translation of the mRNA
• The genetic code is redundant but not
ambiguous
• More than one codon can code for one amino
acid
• No codon specifies more than one amino acid
Third mRNA base (3 end of codon)
First mRNA base (5 end of codon)
Second mRNA base
• The genetic code is universal, shared by
the simplest bacteria to the most complex
animals
• This is why genes can be transcribed and
translated after being transplanted from
one species to another (recombinant DNA
technology)
Pig expressing a jellyfish gene
• Transcription, the first stage of gene
expression, has been examined in great
detail
• RNA synthesis is catalyzed by RNA
polymerase, which pries the DNA strands
apart and hooks together the RNA
nucleotides
• RNA synthesis follows the same base-pairing
rules as DNA, except uracil substitutes for
thymine
• The DNA sequence where RNA
polymerase attaches to a gene is called a
promoter because the presence of this
sequence “promotes” the recognition and
transcription of the gene
• Areas of the DNA lacking promoters are
not transcribed
• The stretch of DNA that is transcribed
is called a transcription unit
Promoter
5
3
Transcription unit
Start point
RNA polymerase
DNA
3
5
Promoter
Transcription unit
5
3
Start point
RNA polymerase
5
3
Unwound
DNA
RNA
transcript
DNA
3
5
1 Initiation
Template strand
of DNA
3
5
Promoter
Transcription unit
5
3
Start point
RNA polymerase
5
3
RNA
transcript
Unwound
DNA
3
5
DNA
1 Initiation
Template strand
of DNA
3
5
2 Elongation
Rewound
DNA
5
3
3
5
RNA
transcript
3
5
Promoter
Transcription unit
5
3
Start point
RNA polymerase
5
3
RNA
transcript
Unwound
DNA
3
5
DNA
1 Initiation
3
5
Template strand
of DNA
2 Elongation
Rewound
DNA
5
3
3
5
3
5
RNA
transcript
3 Termination
5
3
3
5
5
Completed RNA transcript
3
Nontemplate
strand of DNA
Elongation
RNA
polymerase
3
RNA nucleotides
3 end
5
5
Direction of
transcription
(“downstream”)
Newly made
RNA
Template
strand of DNA
• Transcription can be broken down into 3
stages:
– Initiation
– Elongation
– Termination
• Promoters attract proteins called
transcription factors to the gene
• Transcription factors then attract RNA
polymerase so that transcription can be
initiated
• The completed assembly of transcription
factors and RNA polymerase II bound to a
promoter is called a transcription
initiation complex
• Promoters contain A-T rich regions,
making it easier for RNA polymerase to
pry apart (“melt”) the DNA strands
1
Promoter
5
3
A eukaryotic promoter
includes a TATA box
Template
TATA box Start point Template
DNA strand
Transcription
factors
2
Several transcription factors must
bind to the DNA before RNA
polymerase II can do so.
5
3
3
5
3
RNA polymerase II
5
3
3
5
Additional transcription factors bind to
the DNA along with RNA polymerase II,
forming the transcription initiation complex.
Transcription factors
3
5
5
RNA transcript
Transcription initiation complex
• As RNA polymerase moves along the
DNA, it untwists the double helix, 10 to
20 bases at a time
• Transcription progresses at a rate of 40
nucleotides per second in eukaryotes
• A gene can be transcribed simultaneously
by several RNA polymerases
• The mechanisms of termination are
different in bacteria and eukaryotes
• In bacteria, the polymerase stops
transcription at the end of the
terminator
• In eukaryotes, the polymerase continues
transcription after the pre-mRNA is
cleaved from the growing RNA chain; the
polymerase eventually falls off the DNA
• Enzymes in the eukaryotic nucleus modify
pre-mRNA before the genetic messages
are dispatched to the cytoplasm
• During RNA processing, both ends of the
primary transcript are usually altered
• Also, usually some interior parts of the
molecule are cut out, and the other parts
spliced together
• Each end of a pre-mRNA molecule is
modified in a particular way:
– The 5 end receives a modified nucleotide 5
cap
– The 3 end gets a poly-A tail
• These modifications share several
functions:
– They seem to facilitate the export of mRNA
– They protect mRNA from hydrolytic enzymes
– They help ribosomes attach to the 5 end
Structure of a eukaryotic mRNA
G
5
P
P
5’ Cap
P
Protein-coding segment Polyadenylation signal
3
AAUAAA
5’ UTR Start codon
Stop codon
3’ UTR
AAA… AAA
Poly-A tail
• Most eukaryotic genes have long
noncoding stretches of nucleotides that
lie between coding regions
• These noncoding regions are called
intervening sequences, or introns
• The other regions are called exons
because they are eventually expressed,
usually translated into amino acid
sequences
• RNA splicing removes introns and joins
exons, creating an mRNA molecule with a
continuous coding sequence
• Watson and Crick reasoned that the
pairing was more specific, dictated by the
base structures
• They determined that adenine (A) paired
only with thymine (T), and guanine (G)
paired only with cytosine (C)
• The Watson-Crick model explains
Chargaff’s rules: in any organism the
amount of A = T, and the amount of G = C
5’
Pre-mRNA 5’ Cap
Exon Intron
1
30
Exon
31
Coding
segment
mRNA 5’ Cap
1
5’ UTR
Exon
Intron
104
105
3’
146
Poly-A tail
Introns cut out and
exons spliced together
Poly-A tail
146
3’ UTR
• Some genes can encode more than one
kind of polypeptide, depending on which
segments are treated as exons during
RNA splicing
• Such variations are called alternative
RNA splicing
• Because of alternative splicing, the
number of different proteins an organism
can produce is much greater than its
number of genes
• Proteins often have a modular
architecture consisting of discrete
regions called domains
• In many cases, different exons code for
the different domains in a protein
• Exon shuffling may result in the
evolution of new proteins
• Since the two strands of DNA are
complementary, each strand acts as a
template for building a new strand in
replication
• In DNA replication, the parent molecule
unwinds, and two new daughter strands
are built based on base-pairing rules
DNA
Gene
Exon 1 Intron Exon 2 Intron Exon 3
Transcription
RNA processing
Translation
Domain 3
Domain 2
Domain 1
Polypeptide