Microbiology and Immunology

Protein synthesis WORLD OF MICROBIOLOGY AND IMMUNOLOGY

454

•

nisms to route the protein from the Golgi apparatus to the exte-

rior of the cell.

Proteins destined for export in Gram-negative bacteria

are also synthesized as a precursor. The precursor functions at

the outer membrane. Thus, the precursor must cross the inner

membrane intact. This occurs because of an association that

forms between a newly made precursor protein and a complex

of several proteins. The protein complex is referred to as

translocase. The translocase allows the precursor protein, with

the hydrophobic region, to be completely transported across

the inner membrane.

Studies using Escherichia coli and Haemophilus

influenzaedemonstrated the molecular nature of the translo-

case effect. The SecB protein is associated with the precursor

region as a channel running alongside the precursor. The chan-

nel has a hydrophilic and a hydrophobic side. The latter is ori-

ented outward so that it partitions into the hydrophobic

interior of the bacterial inner membrane. The inner surface of

SecB that is in intimate contact with the precursor region is

hydrophilic. Thus, the precursor moves through the inner

membrane in a watery channel.

As the precursor emerges into the periplasm, another

protein present in the periplasm associates with the precursor

region. This association also protects the precursor and

allows the precursor to reach the inner surface of the outer

membrane. Once there, the periplasmic protein is released,

and the precursor sequence spontaneously inserts into the

outer membrane.

Protein export has become an important target of strate-

gies designed to thwart microorganism infections. By block-

ing the ability of certain proteins to be exported, the ability of

bacteria to establish an infection can be hindered.

Conversely, the engineering of proteins to encourage

their export can allow for the easier purification of commer-

cially and clinically important proteins. For example, the

engineering of human insulin in Escherichia colirelies on the

export of the insulin protein. Once free of the bacteria, the

insulin can be recovered in pure form much more easily and

economically than if the protein needed to be extracted from

the bacteria.

See alsoBacteria and bacterial infection; Bacterial mem-

branes and cell wall; Bacterial movement; Bacterial surface

layers; Bacterial ultrastructure; Cell membrane transport;

Enterotoxin and exotoxin; Molecular biology and molecular

genetics; Prokaryotic membrane transport; Proteins and

enzymes

PProtein synthesisROTEIN SYNTHESIS

Protein synthesis represents the final stage in the translation

of genetic information from DNA, via messenger RNA

(mRNA), to protein. It can be viewed as a four-stage process,

consisting of amino acid activation, translation initiation,

chain elongation, and termination. The events are similar in

both prokaryotes, such as bacteria, and higher eukaryotic

organisms, although in the latter there are more factors

involved in the process.

To begin with, each of the 20 cellular amino acids are

combined chemically with a transfer RNA (tRNA) molecule

to create a specific aminoacyl-tRNA for each amino acid. The

process is catalyzed by a group of enzymescalled aminoacyl-

tRNA synthetases, which are highly specific with respect to

the amino acid that they activate. The initiation of translation

starts with the binding of the small subunit of a ribosome,

(30S in prokaryotes, 40S in eukaryotes) to the initiation

codon with the nucleotide sequence AUG, on the mRNA tran-

script. In prokaryotes, a sequence to the left of the AUG

codon is recognized. This is the Shine-Delgrano sequence and

is complementary to part of the small ribosome subunit.

Eukaryotic ribosomesstart with the AUG nearest the 5’-end

of the mRNA, and recognize it by means of a “cap” of 7-

methylguanosine triphosphate. After locating the cap, the

small ribosome subunit moves along the mRNA until it meets

the first AUG codon, where it combines with the large ribo-

somal subunit.

In both prokaryotes and eukaryotes, the initiation com-

plex is prepared for the addition of the large ribosomal sub-

unit at the AUG site, by the release of initiation factor (IF) 3.

In bacteria, the large 50S ribosomal subunit appears simply to

replace IF–3, with IF–1 and IF–2. In eukaryotes, another fac-

tor eIF–5 (eukaryotic initiation factor 5), catalyses the depar-

ture of the previous initiation factors and the joining of the

large 60S ribosomal subunit. In both cases, the release of ini-

tiation factor 2 involves the hydrolysis of the GTP bound to

it. At this stage, the first aminoacyl-tRNA, Met-tRNA, is

bound to the ribosome. The ribosome can accommodate two

tRNA molecules at once. One of these carries the Met-tRNA

at initiation, or the peptide-tRNA complex during elongation

and is thus called the P (peptide) site, while the other accepts

incoming aminoacyl-tRNA and is therefore called the A

(acceptor) site. What binds to the A site is usually a complex

of GTP, elongation factor EF-TU, and aminoacyl-tRNA. The

tRNA is aligned with the next codon on the mRNA, which is

to be read and the elongation factor guides it to the correct

nucleotide triplet. The energy providing GTP is then hydrol-

ysed to GDP and the complex of EF-TU:GDP leaves the ribo-

some. The GDP is released from the complex when the

EF-TU complexes with EF-TS, which is then replaced by

GTP. The recycled EF-TU: GTP is then ready to pick up

another aminoacyl-tRNA for addition to the growing

polypeptide chain. On the ribosome, a reaction is catalysed

between the carboxyl of the P site occupant and the free

amino group of the A site occupant, linking the two together

and promoting the growth of the polypeptide chain. The pep-

tidyl transferase activity which catalyses this transfer is

intrinsic to the ribosome. The final step of elongation is the

movement of the ribosome relative to the mRNA accompa-

nied by the translocation of the peptidyl-tRNA from the A to

the P. Elongation factor EF-G is involved in this step and a

complex of EF-G and GTP binds to the ribosome, GTP being

hydrolysed in the course of the reaction. The de-acylated

tRNA is also released at this time.

womi_P 5/7/03 11:10 AM Page 454