BME 1532-CELL BIOLOGY

Transcription and Translation

by Assist. Prof. Görke Gürel Peközer

Yıldız Technical University

Biomedical Engineering Department
Spring 2020
 Last Week on BME 1532
 Discovery of the DNA as the Hereditary Material
 DNA Structure
 Double Helical Conformation of DNA
 Genome, Gene, Karyotype, Complexity Concepts
 Higher Level DNA Packaging
 Chromatin
 Nucleosomes, Histone Proteins
 Chromosomes
 Heterochromatin-Euchromatin
 DNA Replication
 Semiconservative Nature of DNA Replication
 Replication Forks, Okazaki Fragmens, Primers
 DNA proofreading
 DNA Repair
 Nucleotide Excission Repair
 Mismatch Repair
 Nonhomologous End Joining
 Decoding the Genetic Instructions in DNA
 Once the double-helical structure of DNA had been determined in the early
1950s, it became clear that the hereditary information in cells is encoded in the
linear order—or sequence—of the four different nucleotide subunits that make
up the DNA.
 Genetic instructions written in an alphabet of just four “letters” direct the
formation of the simplest organisms to the most complex ones.
 Cells decode and use the information contained in the genes to direct the
synthesis of proteins.
 Proteins are the principal constituents of cells and determine not only cell
structure but also cell function.
 The properties and function of a protein molecule are determined by the
sequence of the 20 different amino acid subunits in its polypeptide chain: each
type of protein has its own unique amino acid sequence, which dictates how
the chain will fold to form a molecule with a distinctive shape and chemistry.
 The genetic instructions carried by DNA must therefore specify the amino acid
sequences of proteins.
 DNA does not synthesize proteins itself, but it acts
like a manager, delegating the various tasks to a team
of workers.
 When a particular protein is needed by the cell, the
nucleotide sequence of the appropriate segment of a
DNA molecule is first copied into another type of
nucleic acid—RNA (ribonucleic acid ). That segment
of DNA is called a gene, and the resulting RNA
copies are then used to direct the synthesis of the
protein.
 The flow of genetic information in cells is therefore
from DNA to RNA to protein.
 All cells, from bacteria to humans, express their
genetic information in this way—a principle so
fundamental that it has been termed the central
dogma of molecular biology.
 The mechanism by which cells copy DNA into RNA is
called transcription and use the information in
RNA to make protein is called translation.
 Transcription and translation are the means by which cells read out, or
express, the instructions in their genes. Many identical RNA copies can
be made from the same gene, and each RNA molecule can direct the
synthesis of many identical protein molecules.
 This successive amplification enables cells to rapidly synthesize large
amounts of protein whenever necessary. At the same time, each gene
can be transcribed, and its RNA translated, at different rates, providing
the cell with a way to make vast quantities of some proteins and tiny
quantities of others.
 A cell can also regulate the expression of each of its genes according to
the needs of the moment through regulation of gene expression.
 The first step a cell takes in expressing one of its many
thousands of genes is to copy the nucleotide sequence of
that gene into RNA.
 This process is called transcription because the
information, though copied into another chemical form, is
still written in essentially the same language— the
language of nucleotides.
 Like DNA, RNA is a linear polymer made of four different
nucleotide subunits, linked together by phosphodiester
bonds.
 It differs from DNA chemically in two respects:
1. The nucleotides in RNA are ribonucleotides—that is,
they contain the sugar ribose (hence the name
ribonucleic acid) rather than deoxyribose;
2. Although, like DNA, RNA contains the bases adenine
(A), guanine (G), and cytosine (C), it contains uracil (U)
instead of the thymine (T) found in DNA.
 Because U, like T, can base-pair by hydrogen-bonding with
A, the complementary base-pairing properties of DNA
apply also to RNA.
 Although their chemical differences are small, DNA and
RNA differ quite dramatically in overall structure.
 DNA always occurs in cells as a double-stranded helix,
whereas RNA is single-stranded.
 This difference has important functional consequences:
 Because an RNA chain is single stranded, it can fold up into a
variety of shapes, just as a polypeptide chain folds up to form
the final shape of a protein; double stranded DNA cannot fold
in this fashion.
 The ability to fold into a complex three-dimensional shape
allows RNA to carry out various functions in cells, in addition
to conveying information between DNA and protein.
Whereas DNA functions solely as an information store, some
RNAs have structural, regulatory, or catalytic roles.
 All the RNA in a cell is made by transcription, a process that has certain
similarities to DNA replication .
 Transcription begins with the opening and unwinding of a small portion of the
DNA double helix to expose the bases on each DNA strand.
 One of the two strands of the DNA double helix then acts as a template for the
synthesis of RNA. Ribonucleotides are added, one by one, to the growing RNA
chain; as in DNA replication, the nucleotide sequence of the RNA chain is
determined by complementary base-pairing with the DNA template.
 When a good match is made, the incoming ribonucleotide is covalently linked
to the growing RNA chain by the enzyme RNA polymerase.
 The RNA chain produced by transcription—the RNA transcript—is therefore
elongated one nucleotide at a time and has a nucleotide sequence exactly
complementary to the strand of DNA used as the template.
 Transcription differs from DNA replication in several crucial respects:
 Unlike a newly formed DNA strand, the RNA strand does not remain hydrogen-
bonded to the DNA template strand. Instead, just behind the region where the
ribonucleotides are being added, the RNA chain is displaced and the DNA helix
re-forms.
 For this reason—and because only one strand of the DNA molecule is
transcribed—RNA molecules are single-stranded.
 Further, because RNAs are copied from only a limited region of DNA, RNA
molecules are much shorter than DNA molecules.
 Like the DNA polymerase that carries out DNA replication, RNA
polymerases catalyze the formation of the phosphodiester bonds
that link the nucleotides together and form the sugar–phosphate
backbone of the RNA chain.
 The RNA polymerase moves stepwise along the DNA, unwinding
the DNA helix just ahead to expose a new region of the template
strand for complementary base-pairing.
 In this way, the growing RNA chain is extended by one
nucleotide at a time in the 5′-to-3′ direction.
 The incoming ribonucleoside triphosphates (ATP, CTP, UTP, and
GTP) provide the energy needed to drive the reaction forward.
 Although RNA polymerase catalyzes essentially the same
chemical reaction as DNA polymerase, there are some important
differences between the two enzymes.
 First, and most obviously, RNA polymerase uses ribonucleoside for
phosphates as substrates, so it catalyzes the linkage of
ribonucleotides, not deoxyribonucleotides.
 Second, unlike the DNA polymerase involved in DNA replication,
RNA polymerases can start an RNA chain without a primer.
 This difference likely evolved because transcription need not be
as accurate as DNA replication; unlike DNA, RNA is not used as
the permanent storage form of genetic information in cells, so
mistakes in RNA transcripts have relatively minor consequences
for a cell.
 RNA polymerases make about one mistake for every 104
nucleotides copied into RNA, whereas DNA polymerase makes
only one mistake for every 107 nucleotides copied.
 The vast majority of genes carried in a cell’s DNA specify the amino
acid sequences of proteins.
 The RNA molecules encoded by these genes—which ultimately direct
the synthesis of proteins—are called messenger RNAs (mRNAs).
 The final product of other genes, however, is the RNA itself. These
nonmessenger RNAs, like proteins, have various roles, serving as
regulatory, structural, and catalytic components of cells.
 They play key parts, for example, in translating the genetic message
into protein: ribosomal RNAs (rRNAs) form the structural and catalytic
core of the ribosomes, which translate mRNAs into protein, and
transfer RNAs (tRNAs) act as adaptors that select specific amino acids
and hold them in place on a ribosome for their incorporation into
protein. Other small RNAs, called microRNAs (miRNAs), serve as key
regulators of eukaryotic gene expression
 Prokaryotic Transcription
 The initiation of transcription is an especially critical process
because it is the main point at which the cell selects which
proteins or RNAs are to be produced. To begin transcription,
RNA polymerase must be able to recognize the start of a gene
and bind firmly to the DNA at this site.
 The way in which RNA polymerases recognize the transcription
start site of a gene differs somewhat between bacteria and
eukaryotes.
 However, both require a specific sequence of nucleotides called
promoter that lies immediately upstream of the starting point
for RNA synthesis.
 Bacterial trancription is simpler than eukaryotic transcription.
 When a bacterial RNA polymerase collides
randomly with a DNA molecule, the enzyme
sticks weakly to the double helix and then slides
rapidly along its length. RNA polymerase latches
on tightly only after it has encountered the
promoter.
 Once bound tightly to this sequence, the RNA
polymerase opens up the double helix
immediately in front of the promoter to expose
the nucleotides on each strand of a short stretch
of DNA.
 One of the two exposed DNA strands then acts as
a template for complementary base pairing with
incoming ribonucleoside triphosphates, two of
which are joined together by the polymerase to
begin synthesis of the RNA chain.
 Chain elongation then continues until the
enzyme encounters a second signal in the DNA,
the terminator (or stop site), where the
polymerase halts and releases both the DNA
template and the newly made RNA transcript.
 This terminator sequence is contained within the
gene and is transcribed into the 3ʹ end of the
newly made RNA.
 In bacteria, it is a subunit of RNA polymerase, the sigma (σ) factor, that is primarily responsible for
recognizing the promoter sequence on the DNA.
 Each base presents unique features to the outside of the double helix, allowing the sigma factor to
find the promoter sequence without having to separate the DNA strands.
 The next problem an RNA polymerase faces is determining which of the two DNA strands to use as a
template for transcription: each strand has a different nucleotide sequence and would produce a
different RNA transcript.
 The secret lies in the structure of the promoter itself. Every promoter has a certain polarity: it
contains two different nucleotide sequences upstream of the transcriptional start site that position
the RNA polymerase, ensuring that it binds to the promoter in only one orientation.
 Because the polymerase can only synthesize RNA in the 5′-to-3′ direction once the enzyme is bound it
must use the DNA strand oriented in the 3′-to-5′ direction as its template.
 This selection of a template strand does not mean that on a given chromosome, transcription always
proceeds in the same direction. With respect to the chromosome as a whole, the direction of
transcription varies from gene to gene.
 But because each gene typically has only one promoter, the orientation of its promoter determines in
which direction that gene is transcribed and therefore which strand is the template strand.
Bacterial promoters and terminators have specific nucleotide sequences that
are recognized by RNA polymerase.
(A) The green-shaded region represent the nucleotide sequences that specify a promoter. The
numbers above the DNA indicate the positions of nucleotides counting from the first nucleotide
transcribed, which is designated +1. The polarity of the promoter orients the polymerase and
determines which DNA strand is transcribed.
All bacterial promoters contain DNA sequences at –10 and –35 that closely resemble those shown
here. (B) The red-shaded regions represent sequences in the gene that signal the RNA
polymerase to terminate transcription.
Note that the regions transcribed into RNA contain the terminator but not the promoter
nucleotide sequences. By convention, the sequence of a gene is that of the non-template strand,
as this strand has the same sequence as the transcribed RNA (with T substituting for U).
 Eukaryotic Transcription
 Many of the principles we just outlined for bacterial transcription also
apply to eukaryotes. However, transcription initiation in eukaryotes
differs in several important ways from that in bacteria:
 The first difference lies in the RNA polymerases themselves. While
bacteria contain a single type of RNA polymerase, eukaryotic cells have
three—RNA polymerase I, RNA polymerase II, and RNA polymerase III.
These polymerases are responsible for transcribing different types of
genes.
 RNA polymerases I and III transcribe the genes encoding transfer RNA,
ribosomal RNA, and various other RNAs that play structural and
catalytic roles in the cell.
 RNA polymerase II transcribes the vast majority of eukaryotic genes,
including all those that encode proteins and miRNAs.
 A second difference is that, whereas the bacterial RNA polymerase (along with
its sigma subunit) is able to initiate transcription on its own, eukaryotic RNA
polymerases require the assistance of a large set of accessory proteins. Principal
among these are the general transcription factors, which must assemble at each
promoter, along with the polymerase, before the polymerase can begin
transcription.
 A third distinctive feature of transcription in eukaryotes is that the
mechanisms that control its initiation are much more elaborate than those in
prokaryotes.
 In bacteria, genes tend to lie very close to one another in the DNA, with only
very short lengths of nontranscribed DNA between them. But in plants and
animals, including humans, individual genes are spread out along the DNA,
with stretches of up to 100,000 nucleotide pairs between one gene and the
next.
 This architecture allows a single gene to be controlled by a large variety of
regulatory DNA sequences scattered along the DNA, and it enables
eukaryotes to engage in more complex forms of transcriptional regulation than
do bacteria.
 Last but not least, eukaryotic transcription initiation must take into account
the packing of DNA into nucleosomes and more compact forms of chromatin
structure.
 Eukaryotic Transcription
 RNA polymerase II cannot initiate transcription on
its own. It needs general transcription factors.
 These accessory proteins assemble on the promoter,
where they position the RNA polymerase and pull
apart the DNA double helix to expose the template
strand, allowing the polymerase to begin transcription.
 Thus the general transcription factors have a similar
role in eukaryotic transcription as sigma factor has in
bacterial transcription.
 The assembly of general trancription factors at the promoter process
typically begins with the binding of the general transcription factor TFIID
to a short segment of DNA double helix composed primarily of T and A
nucleotides; because of its composition, this part of the promoter is
known as the TATA box.
 Upon binding to DNA, TFIID causes a dramatic local distortion in the
DNA double helix, which helps to serve as a landmark for the subsequent
assembly of other proteins at the promoter.
 The TATA box is a key component of many promoters used by RNA
polymerase II, and it is typically located 25 nucleotides upstream from the
transcription start site.
 Once TFIID has bound to the TATA box, the other factors assemble, along
with RNA polymerase II, to form a complete transcription initiation
complex.
 After RNA polymerase II has been positioned on the promoter, it must be
released from the complex of general transcription factors to begin its task
of making an RNA molecule.
 A key step in liberating the RNA polymerase is the addition of phosphate
groups to its “tail”. This liberation is initiated by the general transcription
factor TFIIH, which contains a protein kinase as one of its subunits.
 Once transcription has begun, most of the general transcription factors
dissociate from the DNA and then are available to initiate another round of
transcription with a new RNA polymerase molecule.
 When RNA polymerase II finishes transcribing a gene, it too is released
from the DNA; the phosphates on its tail are stripped off by protein
phosphatases, and the polymerase is then ready to find a new promoter.
Only the dephosphorylated form of RNA polymerase II can initiate RNA
synthesis.
 Although the templating principle by which DNA is transcribed into RNA is
the same in all organisms, the way in which the RNA transcripts are handled
before they can be used by the cell to make protein differs greatly between
bacteria and eukaryotes.
 Bacterial DNA lies directly exposed to the cytoplasm, which contains the
ribosomes on which protein synthesis takes place. As an mRNA molecule in a
bacterium starts to be synthesized, ribosomes immediately attach to the free 5′
end of the RNA transcript and begin translating it into protein. In eukaryotic
cells, by contrast, DNA is enclosed within the nucleus.
 Transcription takes place in the nucleus, but protein synthesis takes place on
ribosomes in the cytoplasm. So, before a eukaryotic mRNA can be translated
into protein, it must be transported out of the nucleus through small pores in
the nuclear envelope.
 mRNA Processing
 Before it can be exported to the cytosol, however, a
eukaryotic RNA must go through several RNA
processing steps, which include capping, splicing,
and polyadenylation.
 These steps take place as the RNA is being
synthesized. The enzymes responsible for RNA
processing ride on the phosphorylated tail of
eukaryotic RNA polymerase II as it synthesizes an RNA
molecule, and they process the transcript as it emerges
from the polymerase.
 Capping and Polyadenylation
 Different types of RNA are processed in different ways before leaving the nucleus. Two
processing steps, capping and polyadenylation, occur only on RNA transcripts destined
to become mRNA molecules (called precursor mRNAs, or pre-mRNAs).
1. RNA capping modifies the 5′ end of the RNA transcript, the end that is synthesized
first. The RNA is capped by the addition of an atypical nucleotide—a guanine (G)
nucleotide bearing a methyl group, which is attached to the 5′ end of the RNA in an
unusual way . This capping occurs after RNA polymerase II has produced about 25
nucleotides of RNA, long before it has completed transcribing the whole gene.
2. Polyadenylation provides a newly transcribed mRNA with a special structure at its 3′
end. In contrast with bacteria, where the 3′ end of an mRNA is simply the end of the
chain synthesized by the RNA polymerase, the 3′ end of a forming eukaryotic mRNA is
first trimmed by an enzyme that cuts the RNA chain at a particular sequence of
nucleotides. The transcript is then finished off by a second enzyme that adds a series
of repeated adenine (A) nucleotides to the cut end. This poly-A tail is generally a few
hundred nucleotides long
 These two modifications—capping and polyadenylation—increase the stability of a
eukaryotic mRNA molecule, facilitate its export from the nucleus to the cytoplasm, and
generally mark the RNA molecule as an mRNA. They are also used by the protein-
synthesis machinery to make sure that both ends of the mRNA are present and that the
message is therefore complete before protein synthesis begins.
 Introns
 Most eukaryotic pre-mRNAs have to undergo an additional processing
step before they are functional mRNAs. This step involves a far more
radical modification of the pre-mRNA transcript than capping or
polyadenylation, and it is the consequence of a surprising feature of
most eukaryotic genes.
 In bacteria, most proteins are encoded by an uninterrupted stretch of
DNA sequence that is transcribed into an mRNA that, without any
further processing, can be translated into protein.
 Most protein-coding eukaryotic genes, in contrast, have their coding
sequences interrupted by long, noncoding, intervening sequences
called introns. The scattered pieces of coding sequence—called
exons—are usually shorter than the introns, and they often represent
only a small fraction of the total length of the gene.
 To produce an mRNA in a eukaryotic cell, the entire
length of the gene, introns as well as exons, is
transcribed into RNA.
 After capping, and as RNA polymerase II continues
to transcribe the gene, the process of RNA splicing
begins, in which the introns are removed from
the newly synthesized RNA and the exons are
stitched together. Each transcript ultimately
receives a poly-A tail.
 Once a transcript has been spliced and its 5′
and 3′ ends have been modified, the RNA is now
a functional mRNA molecule that can leave the
nucleus and be translated into protein.
 Alternative Splicing
 The intron–exon type of gene arrangement in eukaryotes may, at
first, seem wasteful. It does, however, have a number of
important benefits.
 First, the transcripts of many eukaryotic genes can be spliced in
different ways, each of which can produce a distinct protein. This
process is called alternative splicing and it allows many different
proteins to be produced from the same gene. About 95% of human
genes are thought to undergo alternative splicing. Thus RNA
splicing enables eukaryotes to increase the already enormous
coding potential of their genomes.
 RNA splicing also provides another advantage to eukaryotes, one
that is likely to have been profoundly important in the early
evolutionary history of genes: Novel proteins appear to have arisen
by the mixing and matching of different exons of preexisting genes.
 Eukaryotic pre-mRNA synthesis and processing take place in an orderly fashion within
the cell nucleus.
 Only correctly processed mature mRNA can be transported from the nucleus to the
cytosol where it is translated.
 This selective transport is mediated by nuclear pore complexes, which connect the
nucleoplasm with the cytosol and act as gates that control which macromolecules can
enter or leave the nucleus.
 To be “export ready,” an mRNA molecule must be bound to an appropriate set of proteins,
each of which recognizes different parts of a mature mRNA molecule.
 These proteins include poly-A–binding proteins, a cap-binding complex, and proteins
that bind to mRNAs that have been appropriately spliced.
 The entire set of bound proteins, rather than any single protein, ultimately determines
whether an mRNA molecule will leave the nucleus.
 The “waste RNAs” that remain behind in the nucleus are degraded there, and their
nucleotide building blocks are reused for transcription.
 mRNA life time
 Each mRNA molecule is eventually degraded into nucleotides by
ribonucleases (RNAses) present in the cytosol, but the lifetimes
of mRNA molecules differ considerably—depending on the
nucleotide sequence of the mRNA and the type of cell.
 Because a single mRNA molecule can be translated into protein
many times, the length of time that a mature mRNA molecule
persists in the cell affects the amount of protein it produces.
 Different lifetimes are in part controlled by nucleotide sequences
in the mRNA itself, most often in the portion of RNA called the
3′ untranslated region (UTR), which lies between the 3′ end of
the coding sequence and the poly-A tail.
 The different lifetimes of mRNAs help the cell control the
amount of each protein that it synthesizes.
 Translation
 Transcription as a means of information transfer is simple to
understand: DNA and RNA are chemically and structurally similar, and
DNA can act as a direct template for the synthesis of RNA through
complementary base pairing. The language itself and the form of the
message do not change, and the symbols used are closely related.
 However, the conversion of the information in RNA into protein
represents a translation of the information into another language that
uses different symbols.
 Because there are only 4 different nucleotides in mRNA but 20
different types of amino acids in a protein, this translation cannot be
accounted for by a direct one-to-one correspondence between a
nucleotide in RNA and an amino acid in protein.
 The rules by which the nucleotide sequence of a gene, through an
intermediary mRNA molecule, is translated into the amino acid
sequence of a protein are known as the genetic code.
 The sequence of nucleotides in an mRNA molecule is read
consecutively in groups of three.
 Each group of three consecutive nucleotides in RNA is called a
codon, and each codon specifies one amino acid.
 Because RNA is made of 4 different nucleotides, there are
4 × 4 × 4 = 64 possible combinations of three nucleotides: AAA,
AUA, AUG, and so on.
 However, only 20 different amino acids are commonly found in
proteins.
 Thus, some amino acids are specified by more than one triplet.
 The same genetic code is used in nearly all present-day
organisms. Although a few slight differences have been found,
these occur chiefly in the mRNA of mitochondria and of some
fungi and protozoa.
 Reading Frame
 In the process of translating a nucleotide
sequence (blue) into an amino acid sequence
(red), the sequence of nucleotides in an mRNA
molecule is read from the 5′ to the 3′ end in
sequential sets of three nucleotides.
 In principle, therefore, the same mRNA sequence
can specify three completely different amino acid
sequences, depending on where translation
begins.
 Those three different translation patterns are
called the reading frames.
 An mRNA sequence can be translated in any one
of three different reading frames, depending on
where the decoding process begins.
 However, only one of the three possible reading
frames in an mRNA specifies the correct protein.
 Transfer RNA
 The codons in an mRNA molecule do not directly
recognize the amino acids they specify. Rather, the
translation of mRNA into protein depends on adaptor
molecules that can recognize and bind to a codon at one
site on their surface and to an amino acid at another site.
 These adaptors consist of a set of small RNA molecules
known as transfer RNAs (tRNAs), each about 80
nucleotides in length.
 The base paired regions of tRNA fold back on themselves
to form secondary structures resembling a cloverleaf.
 Two regions of unpaired
nucleotides situated at either
end of the L-shaped tRNA
molecule are crucial to the
function of tRNAs in protein
synthesis.
 One of these regions forms the
anticodon, a set of three
consecutive nucleotides that
bind, through base-pairing, to
the complementary codon in an
mRNA molecule.
 The other is a short single-
stranded region at the 3′ end of
the molecule; this is the site
where the amino acid that
matches the codon is covalently
attached to the tRNA.
 For a tRNA molecule to carry out its role as an adaptor, it must be
linked—or charged—with the correct amino acid.
 Recognition and attachment of the correct amino acid depend on
enzymes called aminoacyl-tRNA synthetases, which covalently couple
each amino acid to its appropriate set of tRNA molecules.
 There are 20 synthetases for 20 aminoacids: one attaches glycine to all
tRNAs that recognize codons for glycine, another attaches
phenylalanine to all tRNAs that recognize codons for phenylalanine,
and so on.
 Each synthetase enzyme recognizes specific nucleotides in both the
anticodon and the aminoacid- accepting arm of the correct tRNA.
 Ribosomes
 The recognition of a codon by the anticodon on a tRNA molecule depends on
the same type of complementary base-pairing used in DNA replication and
transcription.
 However, accurate and rapid translation of mRNA into protein requires a
molecular machine that can move along the mRNA, capture complementary
tRNA molecules, hold the tRNAs in position, and then covalently link the
amino acids that they carry to form a polypeptide chain.
 In both prokaryotes and eukaryotes, the machine that gets the job done is the
ribosome—a large complex made from dozens of small proteins (the ribosomal
proteins) and several crucial RNA molecules called ribosomal RNAs (rRNAs).
 A typical eukaryotic cell contains millions of ribosomes in its cytoplasm.
 Eukaryotic and prokaryotic ribosomes are very
similar in structure and function.
 Both are composed of one large subunit and one
small subunit, which fit together to form a complete
ribosome.
 The small ribosomal subunit matches the
tRNAs to the codons of the mRNA, while the
large subunit catalyzes the formation of the
peptide bonds that covalently link the amino
acids together into a polypeptide chain.
 These two subunits come together on an mRNA
molecule near its 5′ end to start the synthesis of a
protein. The mRNA is then pulled through the
ribosome. As the mRNA moves forward in a 5′-to-3′
direction, the ribosome translates its nucleotide
sequence into an amino acid sequence, one codon
at a time, using the tRNAs as adaptors.
 Each amino acid is thereby added in the correct
sequence to the end of the growing polypeptide
chain. When synthesis of the protein is finished,
the two subunits of the ribosome separate.
 In addition to a binding site for an mRNA
molecule, each ribosome contains three binding
sites for tRNA molecules, called the A site, the P
site, and the E site (short for aminoacyltRNA,
peptidyl-tRNA, and exit, respectively).
 To add an amino acid to a growing peptide
chain, the appropriate charged tRNA enters the
A site by base-pairing with the complementary
codon on the mRNA molecule.
 Its amino acid is then linked to the peptide
chain held by the tRNA in the neighboring P
site. Next, the large ribosomal subunit shifts
forward, moving the spent tRNA to the E site
before ejecting it.
 This cycle of reactions is repeated each time an
amino acid is added to the polypeptide chain,
with the new protein growing from its amino to
its carboxyl end until a stop codon in the mRNA
is encountered.
 In step 1, a charged tRNA carrying the next amino acid to be added
to the polypeptide chain binds to the vacant A site on the ribosome
by forming base pairs with the mRNA codon that is exposed there.
Because only the appropriate tRNA molecules can base-pair with
each codon, this codon determines the specific amino acid added.
 The A and P sites are sufficiently close together that their two tRNA
molecules are forced to form base pairs with codons that are
contiguous, with no stray bases in between. This positioning of the
tRNAs ensures that the correct reading frame will be preserved
throughout the synthesis of the protein.
 In step 2, the carboxyl end of the polypeptide chain (amino acid 3
in step 1) is uncoupled from the tRNA at the P site and joined by a
peptide bond to the free amino group of the amino acid linked to
the tRNA at the A site. This reaction is catalyzed by an enzymatic
site in the large subunit.
 In step 3, a shift of the large subunit relative to the small subunit
moves the two tRNAs into the E and P sites of the large subunit.
 In step 4, the small subunit moves exactly three nucleotides along
the mRNA molecule, bringing it back to its original position
relative to the large subunit.
 This movement ejects the spent tRNA and resets the ribosome with
an empty A site so that the next charged tRNA molecule can bind.
 As indicated, the mRNA is translated in the 5′-to-3′ direction, and
the N-terminal end of a protein is made first, with each cycle
adding one amino acid to the C-terminus of the polypeptide chain.
 Initiation of Translation
 A specific start signal is required to initiate translation.
 The site at which protein synthesis begins on an mRNA is crucial,
because it sets the reading frame for the whole length of the message.
 An error of one nucleotide either way at this stage will cause every
subsequent codon in the mRNA to be misread, resulting in a
nonfunctional protein with an incorrect sequence of aminoacids.
 The translation of an mRNA begins with the codon AUG, and a special
charged tRNA is required to initiate translation.
 This initiator tRNA always carries the amino acid methionine.
 Thus newly made proteins all have methionine as the first amino acid
at their N-terminal end, the end of a protein that is synthesized first.
 This methionine is usually removed later by a specific protease.
 In eukaryotes, an initiator tRNA, charged with methionine, is first
loaded into the P site of the small ribosomal subunit, along with
additional proteins called translation initiation factors.
 The initiator tRNA is distinct from the tRNA that normally carries
methionine.
 Of all the tRNAs in the cell, only a charged initiator tRNA
molecule is capable of binding tightly to the P site in the absence
of the large ribosomal subunit.
 Next, the small ribosomal subunit loaded with the initiator tRNA
binds to the 5′ end of an mRNA molecule, which is marked by the
5′ cap that is present on all eukaryotic mRNAs.
 The small ribosomal subunit then moves forward (5′ to 3′) along
the mRNA searching for the first AUG.
 When this AUG is encountered and recognized by the initiator
tRNA, several initiation factors dissociate from the small
ribosomal subunit to make way for the large ribosomal subunit to
bind and complete ribosomal assembly.
 Because the initiator tRNA is bound to the P site, protein
synthesis is ready to begin with the addition of the next charged
tRNA to the A site.
 End of Translation
 The end of translation in both prokaryotes and eukaryotes is
signaled by the presence of one of several codons, called stop
codons, in the mRNA. The stop codons—UAA, UAG, and
UGA—are not recognized by a tRNA and do not specify an
amino acid, but instead signal to the ribosome to stop
translation.
 Proteins known as release factors bind to any stop codon that
reaches the A site on the ribosome; this binding alters the
activity of the peptidyl transferase in the ribosome, causing it to
catalyze the addition of a water molecule instead of an amino
acid to the peptidyl-tRNA.
 This reaction frees the carboxyl end of the polypeptide chain
from its attachment to a tRNA molecule; because this is the
only attachment that holds the growing polypeptide to the
ribosome, the completed protein chain is immediately released.
 At this point, the ribosome also releases the mRNA and
dissociates into its two separate subunits, which can then
assemble on another mRNA molecule to begin a new round of
protein synthesis.
 Multiple ribosomes usually bind to each
mRNA molecule being translated.
 If the mRNA is being translated efficiently,
a new ribosome hops onto the 5′ end of the
mRNA molecule almost as soon as the
preceding ribosome has translated enough
of the nucleotide sequence to move out of
the way.
 The mRNA molecules being translated are
therefore usually found in the form of
polyribosomes.
 By this way proteins can be synthesized
efficiently in large amounts.