Protein

Synthesis
Kinds of RNA
 The class of RNA found in ribosomes is called
ribosomal RNA (rRNA). During polypeptide
synthesis, rRNA provides the site where polypeptides
are assembled.

 Transfer RNA (tRNA) molecules both transport the

amino acids to the ribosome for use in building the
polypeptides and position each amino acid at the
correct place on the elongating polypeptide chain.
Human cells contain about 45 different kinds of tRNA
molecules.

 Messenger RNA (mRNA) molecules are long

strands of RNA that are transcribed from DNA and
that travel to the ribosomes to direct precisely which
amino acids are assembled into polypeptides.
The Central Dogma
Information passes from
the genes (DNA) to an
RNA copy of the gene,
and the RNA copy
directs the sequential
assembly of a chain of
amino acids
The Genetic Code
 The essential question of gene expression is, “How
does the order of nucleotides in a DNA molecule
encode the information that specifies the order of
amino acids in a polypeptide?”

 The answer came in 1961, through an experiment

led by Francis Crick.

 That experiment was so elegant and the result so

critical to understanding the genetic code that we
will describe it in detail.
Proving code words have only three letters
 Crick and his colleagues reasoned that the genetic
code most likely consisted of a series of blocks of
information called codons.

 They further hypothesized that the information

within one codon was probably a sequence of three
nucleotides specifying a particular amino acid.

 They arrived at the number three, because a two-

nucleotide codon would not yield enough
combinations to code for the 20 different amino acids
that commonly occur in proteins.

 With four DNA nucleotides (G, C, T, and A), only 42,

or 16, different pairs of nucleotides could be formed.
 However, these same nucleotides can be
arranged in 43, or 64, different combinations of
three, more than enough to code for the 20
amino acids.

 When they made a single deletion or two

deletions near each other, the reading frame of
the genetic message shifted, and the
downstream gene was transcribed as
nonsense.

 However, when they made three deletions, the

correct reading frame was restored, and the
sequences downstream were transcribed
correctly.

 They obtained the same results when they

made additions to the DNA consisting of one,
two, or three nucleotides.
The code is practically universal
 For example, the codon AGA specifies the amino acid
arginine in bacteria, in humans, and in all other
organisms whose genetic code has been studied.

 Because the code is universal, genes transcribed

from one organism can be translated in another; the
mRNA is fully able to dictate a functionally active
protein.

 Similarly, genes can be transferred from one

organism to another and be successfully transcribed
and translated in their new host.

 Many commercial products such as the insulin used

to treat diabetes are now manufactured by placing
human genes into bacteria, which then serve as tiny
But Not Quite
 In 1979, investigators began to determine the
complete nucleotide sequences of the mitochondrial
genomes in humans, cattle, and mice.

 It came as something of a shock when these

investigators learned that the genetic code used by
these mammalian mitochondria was not quite the
same as the “universal code” that has become so
familiar to biologists.

 In the mitochondrial genomes, what should have

been a “stop” codon, UGA, was instead read as the
amino acid tryptophan; AUA was read as methionine
rather than isoleucine; and AGA and AGG were read
as “stop” rather than arginine.

 Thus, it appears that the genetic code is not quite

Genes are first transcribed, and then
translated.

Transcription
 The first step in gene expression is the production of
an RNA copy of the DNA sequence encoding the
gene, a process called transcription.

 To understand the mechanism behind the

transcription process, it is useful to focus first on RNA
polymerase, the remarkable enzyme responsible for
carrying it out.
RNA Polymerase
 RNA polymerase is best understood in bacteria.

 Bacterial RNA polymerase is very large and complex,

consisting of five subunits:

1. Two α subunits bind regulatory proteins.

2. β′ subunit binds the DNA template

3. β subunit binds RNA nucleoside subunits.

4. σ subunit recognizes the promoter and initiates

synthesis.
 Only one of the two strands of DNA, called the
template strand, is transcribed.

 The strand of DNA that is not transcribed is called the

coding strand.

 The polymerase adds ribonucleotides to the growing

3′ end of an RNA chain.

 Bacteria contain only one RNA polymerase enzyme,

while eukaryotes have three different RNA
polymerases:

1. RNA polymerase I: synthesizes rRNA in the

nucleolus.
2. RNA polymerase II: synthesizes mRNA.
Promoter
 Transcription starts at RNA polymerase binding
sites called promoters on the DNA template
strand.

 A promoter is a short sequence that is not itself

transcribed by the polymerase that binds to it.

 Promoters differ widely in efficiency.

 Strong promoters cause frequent initiations of

transcription, as often as every 2 seconds in
some bacteria.

 Weak promoters may transcribe only once

every 10 minutes.
Initiation
 In bacteria, a subunit of RNA polymerase called σ
(sigma) recognizes the –10 sequence in the
promoter and binds RNA polymerase there.

 Importantly, this subunit can detect the –10 sequence

without unwinding the DNA double helix.

 In eukaryotes, the –25 sequence plays a similar role

in initiating transcription, as it is the binding site for a
key protein factor.

 Other eukaryotic factors then bind one after another,

assembling a large and complicated transcription
complex.

 Once bound to the promoter, the RNA polymerase

begins to unwind the DNA helix.
Elongation

 Unlike DNA synthesis, a primer is not required.

 The region containing the RNA polymerase, DNA, and

growing RNA transcript is called the transcription
bubble because it contains a locally unwound
“bubble” of DNA.
 The transcription bubble moves down the DNA at a
constant rate, about 50 nucleotides per second,
leaving the growing RNA strand protruding from the
bubble.

 After the transcription bubble passes, the now

transcribed DNA is rewound as it leaves the bubble.

 Unlike DNA polymerase, RNA polymerase has no

proofreading capability.

 Transcription thus produces many more copying

errors than replication.

 Most genes are transcribed many times, so a few

faulty copies are not harmful.
Termination
 At the end of a gene are “stop” sequences that cause
the formation of phosphodiester bonds to cease the
RNA polymerase to release the DNA, and the DNA
within the transcription bubble to rewind.

 The simplest stop signal is a series of GC base-pairs

followed by a series of AT base-pairs.

 The RNA transcript of this stop region forms a GC

hairpin followed by four or more U ribonucleotides.

 How does this structure terminate transcription? The

hairpin causes the RNA polymerase to pause
immediately after the polymerase has synthesized it,
placing the polymerase directly over the run of four
uracils.
 The pairing of U with DNA’s A is the weakest of
the four hybrid base-pairs and is not strong
enough to hold the hybrid strands together
during the long pause.

 Instead, the RNA strand dissociates from the

DNA within the transcription bubble, and
transcription stops.

 A variety of protein factors aid hairpin loops in

Translation
 In prokaryotes, translation begins when the initial
portion of an mRNA molecule binds to an rRNA
molecule in a ribosome.

 The mRNA lies on the ribosome in such a way that

only one of its codons is exposed at the
polypeptidemaking site at any time.

 A tRNA molecule possessing the complementary

three-nucleotide sequence, or anticodon, binds to the
exposed codon on the mRNA.

 Because this tRNA molecule carries a particular

amino acid, that amino acid and no other is added to
the polypeptide in that position.
 As the mRNA molecule moves through the ribosome,
successive codons on the mRNA are exposed, and a
series of tRNA molecules bind one after another to
the exposed codons.

 Each of these tRNA molecules carries an attached

amino acid, which it adds to the end of the growing
polypeptide chain.

 There are about 45 different kinds of tRNA

molecules. Why are there 45 and not 64 tRNAs (one
for each codon)?

 How do particular amino acids become associated

with particular tRNA molecules? The key translation
step, which pairs the three-nucleotide sequences
with appropriate amino acids, is carried out by a
remarkable set of enzymes called activating
enzymes.
Activating Enzymes
 Activating enzymes called aminoacyl-tRNA
synthetases, one of which exists for each of the 20
common amino acids.

 Therefore, these enzymes must correspond to specific

anticodon sequences on a tRNA molecule as well as
particular amino acids.

 Some activating enzymes correspond to only one

anticodon and thus only one tRNA molecule.

 Others recognize two, three, four, or six different

tRNA molecules, each with a different anticodon but
coding for the same amino acid.
“Start” and “Stop” Signals
 There is no tRNA with an anticodon complementary
to three of the 64 codons: UAA, UAG, and UGA.

 These codons, called nonsense codons, serve as

“stop” signals in the mRNA message, marking the
end of a polypeptide.

 The “start” signal that marks the beginning of a

polypeptide within an mRNA message is the codon
AUG, which also encodes the amino acid methionine.

 The ribosome will usually use the first AUG that it

encounters in the mRNA to signal the start of
translation.
Initiation
 Initiation in eukaryotes and prokaryotes is similar,
although it differs in two important ways:

1. First: in eukaryotes, the initiating amino acid is

methionine rather than N-formylmethionine.

2. Second: the initiation complex is far more

complicated than in bacteria, containing nine or more
protein factors, many consisting of several subunits.
Elongation
 When a tRNA molecule with the appropriate
anticodon appears, proteins called elongation factors
assist in binding it to the exposed mRNA codon at the
A site.

 When the second tRNA binds to the ribosome, it

places its amino acid directly adjacent to the initial
methionine, which is still attached to its tRNA
molecule, which in turn is still bound to the ribosome.

 The two amino acids undergo a chemical reaction,

catalyzed by peptidyl transferase, which releases the
initial methionine from its tRNA and attaches it
instead by a peptide bond to the second amino acid.
Translocation
 In a process called translocation the ribosome now
moves (translocates) three more nucleotides along
the mRNA molecule in the 5´ →3´ direction.

 This movement relocates the initial tRNA to the E site

and ejects it from the ribosome, repositions the
growing polypeptide chain to the P site, and exposes
the next codon on the mRNA at the A site.
Termination
 Elongation continues in this fashion until a chain-
terminating nonsense codon is exposed (for example,
UAA).

 Nonsense codons do not bind to tRNA, but they are

recognized by release factors, proteins that release
the newly made polypeptide from the ribosome.
The
End