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Transcription
 Transfer of information from DNA molecule to RNA
molecule.
 DNA  RNA
 Transcription occur in the nucleus for eukaryotes and in
the cytoplasm for prokaryotes.
 Primary product of transcription is three major types of
RNA: mRNA, tRNA, rRNA.
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
Transcription process consist of 3 stages:
1)
2)
3)
INITIATION
ELONGATION
TERMINATION
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INITIATION OF
 Transcription will start at the promoter region located at the
beginning of the gene.
 the enzyme that synthesize RNA is RNA polymerase.
 to initiate transcription, RNA polymerase must first locate the
beginning of the gene.
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 near at the beginning of every gene, there is a region known as
the promoter.
 Transcription will start at this promoter region.
 Promoter is an untranscribed sequence of DNA bases. It usually
consist of one or more repetitions of the sequence TATA.
 when RNA polymerase binds to the promoter region, the DNA
double helix at the beginning of the gene unwinds and
transcription begins.
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ELONGATION OF TRANSCRIPTION PROCEEDS UNTIL RNA
POLYMERASE REACHES A TERMINATION SIGNAL
 the ‘body’ of the gene is where elongation of the RNA strand
occurs.
 RNA polymerase then travels down one of the DNA strands,
called the template strand, synthesizing a single strand of RNA
with bases complementary to those in the DNA.
 base pairing between RNA and DNA is the same as between
two strands of DNA, except that uracil in RNA pairs with
adenine in DNA.
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 after about 10 nucleotides have been added to the growing
RNA chain, the first nucleotides in the RNA molecule separate
from the DNA template strand.
 this separation allows the two dDNA to rewind into a double
helix
 thus, as transcription continues to elongate the RNA molecule,
one end of the RNA drifts away from the DNA; RNA polymerase
keeps the other end temporarily attached to the DNA template
strand.
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 RNA polymerase continues along the template strand of the
gene until it reaches a sequence of the DNA bases known as
termination signal.
 at this point RNA polymerase release the completed RNA
molecules and detaches from the DNA.
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 RNA molecule almost always consists of a single strand.
 In DNA or RNA, the four nucleotide monomers act like the
letters of the alphabet to communicate information.
 To get from DNA, written in one chemical language, to protein,
written in another, requires two major stages, transcription
and translation.
 blocks of three nucleotides (codons), are decoded into a
sequence of amino acids.
 It would take at least 300 nucleotides to code for a
polypeptide that is 100 amino acids long.
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 In prokaryotes, the RNA copy of a gene (mRNA) is ready to be
translated into protein. In fact, translation starts even before
transcription is finished.
 In eukaryotes, the primary RNA transcript of a gene needs further
processing before it can be translated. This step is called “RNA
processing”. Also, it needs to be transported out of the nucleus into
the cytoplasm.
 Steps in RNA processing:
 1. Add a cap to the 5’ end
 2. Add a poly-A tail to the 3’ end
 3. splice out introns.
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 RNA
produced
from
transcription
is
unstable,
especially at the ends. The
ends are modified to protect it.
 At the 5’ end, a slightly
modified guanine (7-methyl G)
is attached “backwards”, by a 5’
to
5’
linkage,
to
the
triphosphates of the first
transcribed base.
 At the 3’ end, the primary
transcript RNA is cut at a
specific site and 100-200
adenine
nucleotides
are
attached: the poly-A tail. These
A’s are not coded in the DNA of
the gene.
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 Introns are regions within a gene that
don’t code for protein and don’t
appear in the final mRNA molecule.
Protein-coding sections of a gene
(called exons) are interrupted by
introns.
 The function
unclear.
of
introns
remains
 There are a few prokaryotic examples,
but most introns are found in
eukaryotes.
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 Introns are removed from the
primary RNA transcript while it
is still in the nucleus.
 Introns are “spliced out” by
RNA/protein hybrids called
“spliceosomes”.
The intron
sequences are removed, and
the remaining ends are reattached so the final RNA
consists of exons only.
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 In eukaryotes, RNA polymerase produces a “primary transcript”, an exact
RNA copy of the gene.
 A cap is put on the 5’ end.
 The RNA is terminated and poly-A is added to the 3’ end.
 All introns are spliced out.
 At this point, the RNA can be called messenger RNA. It is then transported
out of the nucleus into the cytoplasm, where it is translated.
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HOW IS THE BASE SEQUENCE OF A
MESSENGER RNA MOLECULE TRANSLATED
INTO PROTEIN??
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MESSENGER RNA (mRNA) CARRIES THE CODE
FOR PROTEIN SYNTHESIS FROM NUCLEUS TO
THE CYTOPLASMA
 All RNA is produced by transcription of DNA, but only mRNA
carries the code for the amino acid sequence of a protein.
 In the cytoplasm, mRNA binds to ribosomes, which synthesize
a protein specified by the mRNA base sequence.
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Ribosomes consist of two subunits, each composed
of Ribosomal RNA (rRNA) and Protein
 Ribosomes, the structures that carry out translation, are
composed of rRNA and many different proteins.
 Each ribosome is composed of two subunits: small & large.
 The small subunit has binding sites for mRNA, a
‘start’(methionine) tRNA, and several others proteins that
collectively make up the ‘initiation complex’.
 The large subunit has binding sites for two tRNA molecules
and catalytic site for joining together the amino acids attached
to the tRNA molecules.
 During protein synthesis, the small and large subunits come
together and sandwich an mRNA molecules between them.
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Transfer RNA (tRNA) Molecules Decode the Sequence
of Bases in mRNA into the Amino Acid Sequence of a
protein
 The ability of tRNA to deliver the proper amino acid depends on
specific base pairing between tRNA and mRNA.
 Each tRNA has three exposed bases, called – ANTICODON,
which form base pairs with the mRNA codon.
 For example, the mRNA codon AUG forms base pairs with the
anticodon UAC of a tRNA --- that has the amino acid methionine
attached to its end ---incorporate into growing protein.
 If the codon on mRNA is UUU, a tRNA with an AAA anticodon
and carrying phenyalanine will bind to it.
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 tRNA molecules are short RNAs that fold
into a cloverleaf pattern.
 Each tRNA has 3 bases that make up the
anticodon. These bases pair with the 3
bases of the codon on mRNA during
translation.
 Each tRNA has its matching amino acid
attached to the 3’ end. A set of enzymes,
the “aminoacyl tRNA synthetases”, are used
to “charge” the tRNA with the proper amino
acid.
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HOW DOES THE CELL RECOGNIZE WHERE CODONS START
AND STOP?
 All protein originally begin with the same amino acid, methionine – specified by
the codon –AUG (aka start codon).
 Three stop codons: UAG, UAA and UGA will signal the termination of
translation.
 When the ribosome encounters a stop codon, it release both the newly
synthesized protein and the mRNA.
 This establishes the reading frame and subsequent codons are read in groups of
three nucleotides.
 In summary, genetic information is encoded as a sequence of codons, each of
which is translated into a specific amino acid during protein synthesis
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 Each group of 3 nucleotides on the mRNA is a codon. Since there are 4
bases, there are 43 = 64 possible codons, which must code for 20 different
amino acids.
 More than one codon is used for most amino acids (Several codons can
specify the same amino acid): the genetic code is “degenerate”. This
means that it is not possible to take a protein sequence and deduce
exactly the base sequence of the gene it came from.
 AUG is used as the start codon. All proteins are initially translated with
methionine in the first position, although it is often removed after
translation. There are also internal methionines in most proteins, coded
by the same AUG codon.
 There are 3 stop codons, also called “nonsense” codons. Proteins end in
a stop codon, which codes for no amino acid (UAA, UAG, UGA).
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 The genetic code is almost universal.
prokaryotes and eukaryotes.
It is used in both
 However, some variants exist, mostly in mitochondria which
have very few genes.
 For instance, CUA codes for leucine in the universal code, but in
yeast mitochondria it codes for threonine. Similarly, AGA codes
for arginine in the universal code, but in human and
Drosophila mitochondria it is a stop codon.
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TRANSLATION PROCESS
 Three stages of translation :
 1) Initiation of protein synthesis
 2) Elongation of the protein chain
 3) Termination
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Initiation of Translation
 The first AUG codon (codes for methionine) in an mRNA
sequence specifies the start of translation.
 An initiation complex consist of small ribosomal subunit, a
methionine tRNA and several others protein.
 This initiation complex will bind to a molecule of mRNA with
the AUG start codon.
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• Positioning of mRNA is signaled by a ribosome recognition
sequence on the mRNA.
• the initiator tRNA (carrying the methionine amino acid) with the
UAC anticodon will bind to the AUG codon at the mRNA.
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•
•
•
•
Large ribosomal subunit then joins the initiation complex.
Initiator tRNA is in the P site of large ribosomal subunit.
The A site is available for the next tRNA.
Proteins called initiation factors are also required to bring these translation
components together (mRNA, large and small ribosomal subunits and initiator
tRNA).
• The ribosome is now fully assembled and ready to begin translation.
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Elongation of Translation
 The assembled ribosome covers about 30 nucleotides of the
mRNA.
 It holds two mRNA codons in alignment with the two tRNA
binding sites of the large subunit .
 A second tRNA, with an anticodon complementary to the
second codon of the mRNA, moves into the second tRNA
binding site on the large subunit, the A site.
 The amino acids attached to the two tRNAs are now side by
side.
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 The catalytic site of the large subunit breaks the bond holding
the first amino acid (methionine) to its tRNA
 A peptide bond is formed between these two amino acids
 After the peptide bond is formed, the first tRNA in the P site
which is already ‘empty’ is released and the second tRNA now
carries a two-amino acid chain.
 The tRNA in the A site which carries the two-amino acids chain
will translocate to the P site.
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 A new tRNA, with an anticodon complementary to the third
codon of the mRNA, binds to the empty second site (A site).
 The catalytic site on the large subunit now links the third amino
acid onto the growing protein chain.
 The ‘empty’ tRNA leaves the ribosome, the ribosome shifts to
the next codon on the mRNA, and the process repeats, one
codon at a time.
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Termination of Translation
 A stop codon in the mRNA molecule signals the ribosome to terminate
protein synthesis (3 types of stop codons: UAA, UAG, UGA).
 Stop codons do not bind to tRNA, but special proteins bind to the ribosome,
forcing the ribosome to release the finished protein chain and the mRNA.
• Newly synthesized polypeptides frequently undergo posttranslational
modifications to produce the final active form of protein.
• During translation, it is usual for several ribosomes to be bound to the same
mRNA. Such complex are called a polysome.
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An mRNA molecule can be translated in three possible reading
frames.
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Can synthesize 5 to 15 peptide bonds per second.
Most proteins are 100 to 200 amino acids long ---less than a minute.
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MUTATIONS
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Point mutations can affect protein structure and function
 Mutations are changes in the genetic material of a cell (or virus).
 A chemical change in just one base pair of a gene causes a point mutation.
 If these occur in gametes or cells producing gametes, they may be
transmitted to future generations.
 For example, sickle-cell disease is caused by a mutation of a single base pair
in the gene that codes for one of the polypeptides of hemoglobin.
 A change in a single nucleotide from T to A in the DNA template leads to an
abnormal protein.
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 A point mutation that results in the replacement of a pair of
complementary nucleotides with another nucleotide pair is
called a base-pair substitution.
 Some base-pair substitutions have little or no impact on
protein function.
 In silent mutations, alterations of nucleotides still indicate the
same amino acids because of redundancy in the genetic code.
 Other changes lead to switches from one amino acid to
another with similar properties.
 Still other mutations may occur in a region where the exact
amino acid sequence is not essential for function.
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 Changes in amino acids at crucial sites, especially active sites,
are likely to impact function.
 Missense mutations are those that still code for an amino acid
but change the indicated amino acid.
 Nonsense mutations change an amino acid codon into a stop
codon, nearly always leading to a nonfunctional protein.
 Insertions and deletions are additions or losses of nucleotide
pairs in a gene.
 These have a disastrous effect on the resulting protein more
often than substitutions do.
 Unless these mutations occur in multiples of three, they cause
a frameshift mutation.
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 All the nucleotides downstream of the deletion or insertion will
be improperly grouped into codons.
 The result will be extensive missense, ending sooner or later in
nonsense - premature termination.
 Mutations can occur in a number of ways.
 Errors can occur during DNA replication, DNA repair, or DNA
recombination.
 These can lead to base-pair substitutions, insertions, or
deletions, as well as mutations affecting longer stretches of
DNA.
 These are called spontaneous mutations.
 Spontaneous mutation = A mutation occurring in the absence of mutagens, usually
due to errors in the normal functioning of cellular enzymes.
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BASE-PAIR SUBSTITUTION
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BASE-PAIR INSERTION OR DELETION
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