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Transcript
9
The Molecular
Genetics of Gene
Expression
Gene Expression Principles
• Gene expression involves processes of
transcription and translation which result
in the production of proteins whose
structure is determined by genes
• The primary structure of proteins is a
linear sequence of amino acids held
together by peptide bonds
Gene Expression Principles
• Peptide bonds link the carboxyl group of one
amino acid to the amino group of the next
amino acid
• There are twenty naturally
occurring amino acids,
the fundamental building
blocks of proteins
• The linear sequence of
amino acids in proteins is
specified by the coding
information in specific
genes
Gene Expression Principles
• Polypeptide chains are linear polymers of
amino acids
• There are 20 amino acids each with a
unique side chain = R group
• Colinearity: the linear order of amino acids
is encoded in a DNA base sequence
• The base sequence in DNA specifies the
base sequence in RNA transcript
Transcription
• Transcription = production of messenger
RNA (mRNA) complementary to the base
sequence of specific genes
• mRNA differs from DNA in that it is single
stranded, contains ribose sugar instead of
deoxyribose and the
pyrimidine uracil in
place of thymine
RNA Synthesis
• The nucleotide sequence
in the transcribed mRNA
is complementary to the
base sequence in DNA
• RNA is copied from the template strand which
is 3’-to-5’ in the 5’-to-3’ direction =
antiparallela
• RNA synthesis does not require a primer
and proceeds by the addition of nucleotides
to form mRNA chain
RNA Synthesis
• Promoter = nucleotide sequence 5’ to the
transcription start site which is the initial
binding site of RNA polymerase and
transcription initiation factors
• Promoter recognition by RNA polymerase is a
prerequisite for transcription initiation
RNA Synthesis
• Many promoters contain a similar DNA
sequence = TATAAT = “TATA” box (-10) is a
consensus sequence of many promoters
• Consensus promoter sequence at
-35 = TTGACA
• Transcription termination
sites are inverted repeat
sequences which can form
loops in RNA = stop signal
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 = “cap”
Eukaryotic Transcription
• In many eukaryotic genes the coding regions
which specify the structure of proteins are
interrupted by noncoding segments = “split
genes”
• Coding regions = exons
• Noncoding regions
= introns
• Primary transcript
contains exons and
introns; introns are
subsequently
removed = “splicing”
Eukaryotic Transcription
• Additional processing involves the
addition of a series of adenines at the 3’
end of the transcript = “poly A tail”
• The processed transcript contains a 5’ cap
(7-methylguanosine), adjacent exons and
a poly A tail
Eukaryotic Transcription:
Splicing
• RNA splicing occurs in small nuclear
ribonucleoprotein particles (snRNPS) in
spliceosomes
• Consensus sequences
are located at the 5’ end
= donor site and 3’end
= acceptor site of the
intron
• “A” nucleotide from
branch site in intron
attacks “G” at the 5’
• terminus cutting the
RNA which forms a loop
RNA Transcription: Splicing
• Next, the 5’ exon moves to the 3’ splice
acceptor site where a second cut is made by
the spliceosome
• Exon termini are joined and sealed
• The loop is released
as a lariat structure
which is degraded
• The spliced mRNA
contains fused
exons with coding
information only
RNA Transcription: Splicing
• Spliceosomes contain protein and
specialized small RNAs complementary to
the splice junctions to provide specificity
to splicing reaction
• Small nuclear RNAs U1, U2 and U5
recognize splice donor and acceptor sites
by complementary base pairing so that
intron excision is precise
RNA Splicing
Electron micrographs of
a DNA-RNA hybrid
formed from single-strand template DNA
and mRNA show loops of single-strand
DNA corresponding to noncoding intron
regions spliced from mRNA and poly A tail
which is added posttranscriptionally
Translation
• Translation = genetic information encoded
in mRNA specifies the linear sequence of
amino acids in the corresponding protein
• Translation requires mRNA, ribosomes,
transfer RNA (tRNA), aminoacyl tRNA
synthetases, and initiation, elongation and
termination factors
Translation
• mRNA encodes the information which
specifies the primary structure of protein
• Ribosomes are sites of protein synthesis
which contain ribosomal RNA (rRNA) and
protein and are organized in two subunits:
- small subunit = 30S or 40S (density)
- large subunit = 50S or 60S (density)
Translation
• Transfer RNA (tRNA) = adapter molecule
which aligns amino acids in a sequence
specified by mRNA
• Aminoacyl tRNA synthetases = enzymes
which attach amino acids to tRNAs to
form charged tRNAs
• Initiation, elongation and termination
factors = specialized roles in translation
Translation
• Initiation complex =
mRNA + small ribosomal
subunit + tRNA-met
attaches to large subunit
• tRNA-met occupies
P (peptidyl) site
• A second charged tRNA occupies the A
(aminoacyl) site
• Elongation = met is transferred from its tRNA
to amino acid at A site
• Peptide bond links amino acids
Translation
• Once peptide bond is
formed the ribosome
shifts one codon along
the mRNA to the next
codon = translocation,
requires EF-G
• Elongation cycles require EF-Tu-GTP which
uses energy to exchange tRNAs on ribosome
• Peptidyl transferase catalyzes peptide bond
formation
Translation
• tRNAs are covalently attached to specific
amino acids by aminoacyl- synthetases
and contain anti-codon complementary to
the mRNA codon
• Base pairing between the tRNA anti-codon
and the mRNA codon on the ribosome
places amino acids in the correct linear
sequence in translation
Translation
Direction of Synthesis:
• Template strand of DNA
= 3’-to-5’
• mRNA = 5’-to-3’
• polypeptide = amino
terminus (NH2) to carboxy terminus (COOH)
Translation termination:
• No tRNA can bind to stop codon which
causes release of polypeptide
Translation
• Several ribosomes can move in tandem
along a messenger RNA to form
translation unit=polysome
• In prokaryotes a single mRNA may contain
multiple translation initiation sites =
polycistronic mRNA
• Polycistronic mRNAs allow coordinate
regulation of synthesis of more than one
protein
Translation: Genetic Code
• Translation involves the synthesis of
proteins consisting of a chain of amino
acids whose sequence is specified by the
coding information in mRNA
• mRNA carries the “genetic code” =
chemical information originating in DNA
which specifies the primary structure of
proteins
Translation: Genetic Code
Genetic Code:
• Triplet code = three bases in RNA code for
a single amino acid = codon
• There are 64 triplet codons which can be
formed from 4 bases:
- 61 codons specify 20 amino acids;
genetic code is redundant
- 3 are chain terminating “stop” codons
which end translation
Genetic Code
• “AUG” is the initiator codon which
specifies the placement of methionine as
the first amino acid
• 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
Genetic Code
• Genetic evidence for a triplet code comes
from three-base insertions and deletions
• Genetic code specifies a reading frame in
mRNA in which bases are accessed in linear
triplet units
• Frameshift mutations:
alter reading frame by
adding or deleting
non-multiple of three
bases