Proteín Synthesis
Introduction
An mRNA contains a series of codons that interact with
the anticodons of aminoacyl-tRNAs so that a corresponding series of amino acids
is incorporated into a polypeptide chain. The ribosome provides the environment
for controlling the interaction between mRNA and aminoacyltRNA.
The ribosome behaves like a small migrating factory
that travels along the template engaging in rapid cycles of peptide bond
synthesis. Aminoacyl-tRNAs shoot in and out of the particle at a fearsome rate
while depositing amino acids, and elongation factors cyclically associate with
and dissociate from the ribosome.
Together with its accessory factors, the ribosome
provides the full range of activities required for all the steps of protein
synthesis.
FIGURE 8.1 shows the relative dimensions of the components of the
protein synthetic apparatus. The ribosome consists of two subunits that have
specific roles in protein synthesis. Messenger RNA is associated with the small
subunit; ∼30
bases of the mRNA are bound at any time.
The mRNA threads its way along the surface close to
the junction of the subunits. Two tRNA molecules are active in protein
synthesis at any moment, so polypeptide elongation involves reactions taking
place at just two of the (roughly) ten codons covered by the ribosome. The two tRNAs
are inserted into internal sites that stretch across the subunits. A third tRNA
may remain on the ribosome after it has been used in protein synthesis before
being recycled.
The basic form of the ribosome has been conserved in
evolution, but there are appreciable variations in the overall size and
proportions of RNA and protein in the ribosomes of bacteria, eukaryotic
cytoplasm, and organelles.
FIGURE 8.2 compares the components of bacterial and mammalian
ribosomes. Both are
ribonucleoprotein particles that contain more RNA than
protein. The ribosomal proteins are known as r-proteins.
Each of the ribosome subunits contains a major rRNA
and a number of small proteins.
The large subunit may also contain smaller RNA(s). In E.
coli, the small (30S) subunit consists of the 16S rRNA and 21 r-proteins.
The large (50S) subunit contains 23S rRNA, the small 5S RNA, and 31 proteins.
With the exception of one protein present at four copies per ribosome, there is
one copy of each protein. The major RNAs constitute the major part of the mass
of the bacterial ribosome. Their presence is pervasive, and probably most or
all of the ribosomal proteins actually contact rRNA. So the major rRNAs form
what is sometimes thought of as the backbone of each subunit—a continuous
thread whose presence dominates the structure and which determines the
positions of the ribosomal proteins.
The ribosomes of higher eukaryotic cytoplasm are
larger than those of bacteria. The total content of both RNA and protein is
greater; the major RNA molecules are longer (called 18S and 28S rRNAs), and
there are more proteins.
Probably most or all of the proteins are present in
stoichiometric amounts. RNA is still the predominant component by mass. Organelle
ribosomes are distinct from the ribosomes of the cytosol and take varied forms.
In some cases, they are almost the size of bacterial ribosomes and have 70%
RNA; in other cases, they are only 60S and have <30% RNA.
The ribosome possesses several active centers, each of
which is constructed from a group of proteins associated with a region of
ribosomal RNA. The active centers require the direct participation of rRNA in a
structural or even catalytic role. Some catalytic functions require individual
proteins, but none of the activities can be reproduced by isolated proteins or
groups of proteins; they function only in the context of the ribosome.
Two types of information are important in analyzing the
ribosome. Mutations implicate particular ribosomal proteins or bases in rRNA in
participating in particular reactions.
Structural analysis, including direct modification of components
of the ribosome and comparisons to identify conserved features in rRNA,
identifies the physical locations of components involved in particular
functions.
Protein Synthesis Occurs by Initiation, Elongation, and
Termination
An amino acid is brought to the ribosome by an
aminoacyl-tRNA. Its addition to the growing protein chain occurs by an
interaction with the tRNA that brought the previous amino acid.
Key concepts:
• The ribosome has three tRNA-binding sites.
• An aminoacyl-tRNA enters the A site.
• Peptidyl-tRNA is bound in the P site.
• Deacylated tRNA exits via the E site.
• An amino acid is added to the polypeptide chain by transferring
the polypeptide from peptidyl-tRNA in the P site to aminoacyl-tRNA in the A
site.
FIGURE 8.2 Ribosomes are large ribonucleoprotein particles that
contain more RNA than protein and dissociate into large and small subunits.
Each of these tRNA lies in a distinct site on the ribosome.
FIGURE 8.3 shows that the two sites have different features:
• An incoming aminoacyl-tRNA binds to the A site. Prior
to the entry of aminoacyl tRNA, the site exposes the codon representing the
next amino acid due to be added to the chain.
• The codon representing the most recent amino acid to
have been added to the nascent polypeptide chain lies in the P site. This
site is occupied by peptidyltRNA, a tRNA carrying the nascent polypeptide
chain.
FIGURE 8.4 shows that the aminoacyl end of the tRNA is located on
the large subunit, whereas the anticodon at the other end interacts with the
mRNA bound by the small subunit.
So the P and A sites each extend across both ribosomal
subunits. For a ribosome to synthesize a peptide bond, it must be in the state
shown in step 1 in Figure 8.3, when peptidyl-tRNA is in the P site and aminoacyl-tRNA
is in the A site. Peptide bond formation occurs when the polypeptide carried by
the peptidyl-tRNA is transferred to the aminoacid carried by the
aminoacyl-tRNA. This reaction is catalyzed by the large subunit of the
ribosome.
Transfer of the polypeptide generates the ribosome
shown in step 2, in which the deacylated tRNA, lacking any amino acid,
lies in the P site and a new peptidyl-tRNA has been created in the A site. This
peptidyl-tRNA is one amino acid residue longer than the peptidyltRNA that had
been in the P site in step 1. The ribosome now moves one triplet along the
messenger. This stage is called translocation.
The movement transfers the deacylated tRNA out of the
P site and moves the peptidyltRNA into the P site (see step 3 in the figure). The
next codon to be translated now lies in the A site, ready for a new
aminoacyl-tRNA to enter, when the cycle will be repeated.
FIGURE 8.5 summarizes the interaction between tRNAs and the ribosome.
The deacylated tRNA leaves the ribosome via another tRNA-binding site, the E
site. This site is transiently occupied by the tRNA en route between leaving
the P site and being released from the ribosome into the cytosol. Thus the flow
of tRNA is into the A site, through the P site, and out through the E site (see
also Figure 8.28 in Section 8.12).
FIGURE 8.6 compares the movement of tRNA and mRNA, which may be
thought of as a sort of ratchet in which the reaction is driven by the
codon–anticodon interaction.
Protein synthesis falls into the three stages shown in
FIGURE 8.7:
• Initiation
involves the reactions that precede formation of the peptide bond between the
first two amino acids of the protein. It requires the ribosome to bind to the
mRNA, which forms an initiation complex that contains the first aminoacyl-tRNA.
This is a relatively slow step in protein synthesis and usually determines the
rate at which an mRNA is translated.
• Elongation includes all the reactions from
synthesis of the first peptide bond to addition of the last amino acid. Amino
acids are added to the chain one at a time; the addition of an amino acid is
the most rapid step in protein synthesis.
• Termination encompasses the steps that are
needed to release the completed polypeptide
chain; at the same time, the ribosome dissociates from the mRNA.
Different sets of accessory factors assist the
ribosome at each stage. Energy is provided at various stages by the hydrolysis
of guanine triphosphate (GTP).
During initiation, the small ribosomal subunit binds
to mRNA and then is joined by the 50S subunit. During elongation, the mRNA
moves through the ribosome and is translated in triplets. (Although we usually
talk about the ribosome moving along mRNA, it is more realistic to think in
terms of the mRNA being pulled through the ribosome.) At termination the
protein is released, mRNA is released, and the individual ribosomal subunits
dissociate in order to be used again.
