Transcript Chapter08_Outline

Chapter 8
The Molecular Genetics of
Gene Expression
Gene Expression Steps
• Gene expression is the process by which information
contained in genes is decoded to produce other
molecules that determine the phenotypic traits of
organisms
The principal steps in gene expression are:
• Transcription: RNA molecules are synthesized by an
enzyme, RNA polymerase, which uses a segment of a
single strand of DNA as a template strand to produce
a strand of RNA complementary in base sequence to
the template DNA
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Gene Expression Steps
• Processing: in the nucleus of eukaryotic cells, the
RNA usually undergoes chemical modification
• Translation: the processed RNA molecule is used to
specify the order in which amino acids are joined
together to form a polypeptide chain. In this manner,
the amino acid sequence in a polypeptide is a direct
consequence of the base sequence in the DNA
• The protein made is called the gene product
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Polypeptides
• Polypeptide chains are linear polymers of amino
acids
• There are twenty naturally occurring amino acids,
the fundamental building blocks of proteins
• Peptide bonds link the carboxyl group of one amino
acid to the amino group of the next amino acid
• The sequence of amino acids in proteins is specified
by the coding information in specific genes
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Figure 8.1: The general structure of an amino acid
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Figure 8.3: Properties of a polypeptide chain
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Protein Domains
• Most polypeptides include regions that can fold in
upon themselves to acquire well-defined structures
– domains
• Domains interact with each other and often have
specialized functions
• Individual domains in a protein usually have
independent evolutionary origins; they come
together in various combinations to create genes
with novel functions via duplication of their coding
regions and genomic rearrangements
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Protein Domains
• Domains can be identified through computer
analysis of the amino acid sequence
• Vertebrate genomes have relatively few proteins
or protein domains not found in other organisms.
Their complexity arises in part from innovations
in bringing together preexisting domains to create
novel proteins that have more complex domain
architectures than those found in other
organisms.
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Colinearity
• The linear order of nucleotides in a gene determines
the linear order of amino acids in a polypeptide
• This attribute of genes and polypeptides is called
colinearity, which means that the sequence of base
pairs in DNA determines the sequence of amino acids
in the polypeptide in a colinear, or point-to-point,
manner
• Colinearity is universally found in prokaryotes
• In eukaryotes, noninformational DNA sequences
interrupt the continuity of most genes
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Transcription
• Transcription is the
process of synthesis of an
RNA molecule copied from
the segment of DNA that
constitutes the gene
• RNA differs from DNA in
that it is single stranded,
contains ribose sugar
instead of deoxyribose
and the pyrimidine uracil
in place of thymine
Figure 8.5: Differences between the
structures
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RNA Synthesis
• The nucleotide sequence in the transcribed mRNA is
complementary to the base sequence in DNA
• In the synthesis of RNA, a sugar–phosphate bond is
formed between the 3'- hydroxyl group of one
nucleotide and the 5'- OH triphosphate of the next
nucleotide in line
• RNA synthesis does not require a primer
• The enzyme used in transcription is RNA polymerase
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Figure 8.6A: Base pairing with the template strand
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Figure 8.6B: The polymerization step
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Figure 8.6C: Geometry of RNA synthesis
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RNA Polymerases
• RNA polymerases are large, multisubunit complexes
whose active form is called the RNA polymerase
holoenzyme
• Bacterial cells have only one RNA polymerase
holoenzyme, which contains six polypeptide chains
Figure 8.7: Subunit structure of RNA
polymerase from T. aquaticus
Reprinted from Cell., vol. 98, R. Mooney and R. Landick, " RNA
Polymerase Unveiled", pg. 4, copyright (1999), with permission from
Elsevier [http://www.sciencedirect.com/science/journal/00928674]. 15
RNA Polymerases
•Eukaryotes have several types of RNA polymerase:
•RNA polymerase I transcribes ribosomal RNA.
•RNA polymerase II–all protein-coding genes as well
as the genes for small nuclear RNAs
•RNA polymerase III–tRNA genes and the 5S
component of rRNA
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RNA Synthesis
• Particular nucleotide sequences define the beginning
and end of a gene
• Promoter: nucleotide sequence, 20–200 bp long, is the
initial binding site of RNA polymerase and
transcription initiation factors
• Promoter recognition by RNA polymerase is a
prerequisite for transcription initiation
Figure 8.9: Base sequences in promoter regions of several genes in E. coli
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RNA Synthesis
• Promoter sequences in eukaryotes are generally
much longer and more complex than those in
prokaryotes.
Figure 8.10: Human TATA-box promoter showing sequences in the core
promoter region
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RNA Synthesis
• The consensus promoter regions in E. coli are:
TTGACA (-35), centered approximately 35 base pairs
upstream from the transcription start site (numbered
the +1 site)
TATAAT (-10) = “TATA” box: 10 base pairs upstream
• Transcription termination sites are inverted repeat
sequences that can form loops in RNA: stop signal
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Figure 8.13: Transcription termination
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Eukaryotic Transcription
• Eukaryotic transcription involves the synthesis of
RNA specified by DNA template strand to form a
primary transcript
• Primary transcript is processed to form mRNA that is
transported to the cytoplasm
• The first processing step adds 7-methylguanosine
to the 5'-end of the primary transcript, cap
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Eukaryotic Transcription
• Translation of an mRNA molecule rarely starts
exactly at one end and proceeds to the other end:
initiation of protein synthesis may begin many
nucleotides downstream from the 5'-end
• The 5'untranslated region followed by an open
reading frame (ORF), which specifies polypeptide
chain
• In many eukaryotic genes ORFs are interrupted by
noncoding segments, introns
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Eukaryotic Transcription
• Primary transcript contains exons and introns;
introns are subsequently removed by splicing
• The 3'-end of an mRNA molecule following the ORF
also is not translated; it is called the 3’ untranslated
region
• The 3'- end is usually modified by the addition
of a sequence called the poly-A tail
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Figure 8.16: In eukaryotes, transcription and RNA processing are coupled
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Splicing
• RNA splicing occurs in nuclear particles known
as spliceosomes
• The specificity of splicing comes from the five
small snRNP—RNAs denoted U1, U2, U4, U5, and
U6, which contain sequences complementary to
the splice junctions
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Figure 8.17: Splicing
Adapted from H. D. Madhani and C. Guthrie, Annu. Rev. Genet. 1 (1994):
1-26.
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Figure 8.18A: An electron micrograph of a DNA–RNA hybrid
Courtesy of Thomas R. Broker and Louise T. Chow, University of
Alabama at Birmingham. Original research completed in 1977 at the Cold
Spring Harbor Laboratory, New York.
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Figure 8.18B: RNA and DNA strands
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Splicing
• Human genes tend to be very long even though
they encode proteins of modest size
• The average human gene occupies 27 kb of
genomic DNA, yet only 1.3 kb (~ 5 %) is used to
encode amino acids
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Splicing
• The correlation between exons and domains
found in some genes suggests that the genes
were originally assembled from smaller pieces
• The model of protein evolution through the
combination of different exons is called the exon
shuffle model
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Translation
• The synthesis of every protein molecule in a cell is
directed by an mRNA originally copied from DNA
• Protein production includes two kinds of
processes:
1. information-transfer processes, in which the
RNA base sequence determines an amino acid
sequence
2. chemical processes, in which the amino
acids are linked together.
• The complete series of events is called translation
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Translation
• The translation system consists of five major
components:
 Messenger RNA: mRNA is needed to provide the coding
sequence of bases that determines the amino acid sequence
in the resulting polypeptide chain
 Ribosomes are particles on which protein synthesis takes
place
 Transfer RNA: tRNA is a small adaptor molecule that
translates codons into amino acid
 Aminoacyl-tRNA synthetases: set of molecules catalyzes the
attachment of a particular amino acid to its corresponding
tRNA molecule
 Initiation, elongation, and termination factors
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Translation: Initiation
• In eukaryotes, initiation takes place by scanning the
mRNA for an initiation codon
• In the translation initiation, the 5' cap on the mRNA is
instrumental
• The elongation factor eIF4F binds to the cap
and recruits eIF4A and eIF4B
• This creates a binding site for a charged tRNAMet (an
initiator tRNA), bound with elongation factor eIF2, and
a small 40S ribosomal subunit together with eIF3 and
eIF5
• These components all come together at the 5' cap and
form the 48S initiation complex
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Figure 8.19a/b: Initiation of protein synthesis
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Translation: Initiation
• The initiation complex moves along the mRNA in the 3'
direction, scanning for the first of the initial methionine
codon AUG
• At this point eIF5 causes the release of all the initiation
factors and the recruitment of a large 60S ribosomal subunit
• This subunit includes three binding sites for tRNA molecules:
the E (exit) site, the P (peptidyl) site, and the A (aminoacyl)
site.
• At the beginning the tRNAMet is located in the P site and the A
site is the next in line to be occupied.
• The tRNA binding is accomplished by hydrogen bonding
between the AUG codon in the mRNA and the three-base
anticodon in the tRNA.
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Figure 8.19b/c: Initiation of protein synthesis
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Translation: Elongation
• In the first step of elongation, the 40S subunit moves
one codon farther along the mRNA, and the charged
tRNA corresponding to the new codon is brought into
the A site on the 60S subunit
• A peptidyl transferase activity catalyzes a coupled
reaction in which the bond connecting the
methionine to the tRNAMet is transferred to the amino
group of the next amino acid, forming the first
peptide bond
• In the next step, the 60S subunit swings forward to
catch up with the 40S, and at the same time the
tRNAs in the P and A sites of the large subunit are
shifted to the E and P sites, respectively
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Figure 8.20a/b: Elongation cycle in protein synthesis
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Figure 8.20c/d: Elongation cycle in protein synthesis
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Translation: Elongation
• One cycle of elongation is now completed, and the
entire procedure is repeated for the next codon
• Eukaryotes synthesize a polypeptide chain at the
rate of about 15 amino acids per second
• Elongation in prokaryotes is a little faster (about 20
amino acids per second), but the essential
processes are very similar
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Translation: Termination
• When a stop codon is encountered, the tRNA
holding the polypeptide remains in the P site, and a
release factor (RF) binds with the ribosome.
• GTP hydrolysis provides the energy to cleave the
polypeptide from the tRNA to which it is attached
• The 40S and 60S subunits are recycled to initiate
translation of another mRNA
• Eukaryotes have only one release factor that
recognizes all three stop codons: UAA, UAG, and
UGA
• There are three release factors in E. coli
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Figure 8.21: Termination of protein synthesis
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Translation
• The mRNA is translated in the 5' -to-3' direction.
The polypeptide is synthesized from the amino
end toward the carboxyl end
Figure 8.26: Direction of synthesis of RNA
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Translation
• Most polypeptide chains fold correctly as they exit
the ribosome: they pass through a tunnel in the
large ribosomal subunit that is long enough to
include about 35 amino acids
• Emerging from the tunnel, protein enters into a sort
of cradle formed by a protein associated with the
ribosome: it provides a space where the
polypeptide is able to undergo its folding process.
• The proper folding of more complex polypeptides
is aided by proteins called chaperones and
chaperonins
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Figure 8.24: Alternative pathways in protein folding
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Translation: Prokaryotes
• In prokaryotes, mRNA molecules have no cap and
there is no scanning mechanism
• In E. coli, IF-1 and IF-3 initiation factors interact with
the 30S subunit and IF-2 binds with a special tRNA
charged with formylmethionine tRNAfMet
• These components bind with an mRNA at the
ribosome-binding site, RBS or the Shine–Dalgarno
sequence. Together, they recruit a 50S sub-unit
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Translation: Prokaryotes
• mRNA molecules contain information for the
amino acid sequences of several different
proteins; such a molecule is called a polycistronic
mRNA
• Cistron: DNA sequence that encodes a single
polypeptide chain
• In a polycistronic mRNA, each protein coding
region is preceded by its own ribosome-binding
site and AUG initiation codon
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Translation: Prokaryotes
• After the synthesis of one polypeptide is finished,
the next along the way is translated
• The genes contained in a polycistronic mRNA often
encode the different proteins of a metabolic
pathway.
Figure 8.25: Different products are translated from a threecistron mRNA molecule by
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the ribosomes of prokaryotes and eukaryotes
Genetic Code
• The genetic code is the list of all codons and
the amino acids that they encode
• Main features of the genetic code were proved
in genetic experiments carried out by F.Crick
and collaborators:
• Translation starts from a fixed point
• There is a single reading frame maintained
throughout the process of translation
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Genetic Code
• Each codon consists of three nucleotides. Evidence
for a triplet code came from three-base insertions and
deletions.
• Code is nonoverlapping
• Code is degenerate: each amino acid is specified by
more than one codon
• Most of the codons were determined from in vitro
polypeptide synthesis
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Mutations that delete or add a base pair shift the
reading frame and are called frameshift mutations.
Figure 8.28: Shift in the reading frame
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Figure 8.29: Interpretation of the rII frameshift mutations
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Genetic Code
• Genetic code is universal:
the same triplet codons
specify the same amino
acids in all species
• Mutations occur when
changes in codons alter
amino acids in proteins
Table 8.3: The Standard Genetic
Code
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