Transcript Ch. 10: Presentation Slides

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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
• In the nucleus of eukaryotic cells, the RNA usually
undergoes chemical modification called RNA
processing
• 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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Fig. 8.3
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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
• The domains often have specialized functions
• The 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 = 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
Fig. 8.5
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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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Fig. 8.6a,b
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Fig. 8.6c
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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
• 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
Fig. 8.8
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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 which can form loops in RNA = stop
signal
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Fig. 8.9
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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 which
is transported to the cytoplasm
• The first processing step adds 7- methylguanosine
to 5’-end of the primary transcript = cap
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Fig. 8.12
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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
• Coding regions interrupted by introns—exons
• 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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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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Fig. 8.13
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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
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:
• information-transfer processes, in which the RNA
base sequence determines an amino acid
sequence
• 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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Fig. 8.15a, b
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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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Fig. 8.15b, c
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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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Fig. 8.16a, b
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Fig. 8.16c, d
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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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Translation
• The mRNA is translated in the 5’-to-3’ direction. The
polypeptide is synthesized from the amino end toward the
carboxyl end
• 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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Fig. 8.22
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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
• mRNA molecules contain information for the amino
acid sequences of several different proteins; such a
molecule is called a polycistronic mRNA
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Translation: Prokaryotes
• 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
• 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
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pathway.
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
• Each codon consists of three nucleotides
• Code is nonoverlapping
• Code is degenerate: each amino acid is specified by
more than one codon
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Fig. 8.24
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Genetic Code
• Most of the codons were
determined from in
vitropolypeptide
synthesis
• 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
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