Fundamental concepts and definitions

Protein synthesis is the level of gene expression where genetic information camed as a
messenger RNA (mRNA) molecule is translated into a polypeptide.
The components of the protein synthesis machinery arem RNA, the template which contains
the code to be translated, ribosomes, large ribonucleoprotein particles which are the sites of
protein synthesis, transfeRr NA (tRNA), versatilea daptor molecules which carry amino acids
to the ribosome and facilitate the proceofs st ranslation, and accessory factors associating transiently
with the ribosome, required for ribosome assembly and disassembly, and its activity
during elongation.
Like other polymerization reactions, protein synthesis has stagofe sin itiation, elongation and
termination, each of which may be regulated. The protein synthesis mechanism is similar in
prokaryotes and eukaryotes, but there are subtle differences in the natuorf et he components
and their order of assembly. There are major differences, however, concerning the context in
which protein synthesis occurs. In bacteria, transcription and protein syntohcecsuisr simultaneously
in the cytoplasm (allowing cross-regulation between different levels of gene expression)
and mRNA may be polycistronic. In eukaryotes, transcription is confined to the nucleus
and the mRNA is exported to the cytoplasm for translation. Nascent mRNA is extensively
processed before export andis usually monocistronic (see RNA Processing).
Following synthesis, a polypeptide undergoes further processing before becoming active. It
must fold correctly,a process sometimes requiring the assistancoef a molecular chaperone (q.v.).
It may be cleaved, and specific residues may be chemically modified or conjugated to small
. molecules. Such modifications are often associated with the targeting of proteins to specific
compartments in the cell or for secretion. Proteins may need to associate noncovalently with
other proteins or with nonpolypeptide cofactors for ftuhlel iarc tivity. Fora discussion of these
processes, see Proteins: Structure, function and evolution.
23.1 The components of protein synthesis
Messenger RNA. Messenger RNA (mRNA) is the template for protein synthesis. It has two essential
features: an open reading frame (a sequence of translatable codons) and a ribosome binding
site (where the small ribosome subunit binds and the rest of the ribosome assembles). There are
important distinctions between prokaryotes and eukaryotes with respect to the organization of
these sites, and also concerning other aspects of the life of a typical mRNA molecule. These differences
and their consequences are summarized below.
(1) In bacteria, transcription and translation occur simultaneously in the same cellular compartment,
whereas in eukaryotes, transcription is restricted to the nucleus and RNA must be exported
into the cytoplasm for translation.
(2) Bacterial mRNA has a limited half-life (severaml inutes for the most stable transcripts). Some
eukaryote mRNAs are also unstable, but most are stable for hours or even days (e.g. in eggs).
(3) Bacterial transcripts are used directly for translation, whereas eukaryotic transcripts are
extensively processeda nd modified beforehand. Processing reactionisn clude the splicing of introns
and 3' end polyadenylation; both may regulate the efficiency of translation, either directly or by
modulating mRNA stability. A further modification is the synthesis of a 5' end 7-methylguanosine
cap, which has a direct role in ribosome binding. Some transcripts are also edited (q.v. RNA editing).
314 Advanced Molecular Biology
(4) Ribosome binding in bacteria depends on a conserved motif in the mRNA which complements
part Of the ribosomal 16s rRNA. Binding occurs wherever this sequence is found, including
internally, allowing bacterial transcripts to be polycistronic. Eukaryotic ribosomes are docked onto
the mRNA by a protein which recognizes the modified 5' cap. Thereforeb inding cannoto ccur internally,
and eukaryotic mRNAs are almost universally monocistronic (some RNA viruses, however,
have managed to circumvent this restriction in order to express genes located on their own - polycistronic
- genomes; q.v. infernal ribosome entry site). The structures of typical bacterial and eukaryotic
mRNAs are shown in Figure 8.2 (see The Gene).
The broad consequences of these differencaerse reflected in the regulatory strategies used byb atteria
and eukaryotes. Bacterial mRNA is transcribed, translated and degraded in quick succession,
and the close association of these three processes allows a significant amount of cross-regulation
between different levels of gene expres(sqi.ovn. a ttenuation, refroregulation).C onversely, in eukaryotes
there is a considerable delay between transcription and translation while the mRNA is first processed
and then exported from the nucleus. Both of thesee vents are potential targets for regulation.
Ribosomes. Ribosomes are large, abundant ribonucleoprotein complexes upon which protein SFthesis
occurs. They are found free in the cytoplasm and, in eukaryotes, associated with the membrane
of the rough endoplasmicr eticulum. Ribosomes mayf unction alone (monosomes) although
it is common to see them in clusters concurrently translating the same mRNA (polysomes).
Polysomes can bee xtracted from cellsa nd used to purify mRNA (q.v. poly(A)+ RNA).
All ribosomes comprise two dissimilar sized subunits, the large and small subunits. Each subunit
consists of several ribosomal RNAs (rRNAs) and numerous ribosomal proteins (r-proteins).
Their relative sizes are often expressed in Svedberg units (Box 23.2). In E. coli, the 70s ribosome is
composed of a small 30s subunit and a large 50s subunit. The small subunit contains 21 different
proteins (named Sl-S21), and the 16s rRNA. The large subunit comprises 34 proteins (named
Ll-L34) and the 23s and 5s rRNAs. Some proteins are common to both subunits (e.g. L6 = S20).
Eukaryote ribosomes are larger (80s) and contain more components. The small (40S)'subunit comprises
33 proteins and the 18s rRNA whilst the large (60s) subunit contains 50 proteins and three
rRNAs of 28S,5.8S and 5s. The 5.8s rRNA is homologous to the 5' portion of the bacterial 23s
rRNA
and forms complementary pairs with the equivalent eukaryotic 28s rRNA. Archaen ribosomes
resemble those of bacteria, but some genera contain extra subunits similar to those of eukaryotes.
The spatial organization of the ribosome is complex. rRNA makesu p 6045% of the total mass
and is essential for structural integrity and function, adopting complex tertiary and quaternary conformations
by intra- and intermolecular base pairing. The manner in which proteins interact with
the RNA and each other within this network hasb een studied in great detail for the E. coli ribosome
using cross-linking and footprinting studies, and structural analysis by neutron scattering and diffraction.
Although the primary nucleic acid sequences of rRNAs from different species vary considerably,
it appears that most of the secondary structures are conserved, suggesting that all ribosomes
share a common organization.
Several domains of the ribosome have particularly important functions during protein synthesis.
The small subunit contains a binding site for mRNA and two major binding sites for tRNA. The
A-site (amino acyl-tRNA site) binds incoming charged tRNAsd uring elongation, whilst the P-site
(peptidyl-tRNA site) binds the tRNA carrying the nascent polypeptide chain. Bacterial ribosomes
possess at hird E-site (exit site) to whichs pent tRNAs ared ispatched prior to ejection. The largesu bunit
possesses a petidyltransferase domain, which provides the catalytic activity for peptide bond
formation, and a GTPase domain, whose activities are required for translocation of the ribosome
along the mRNA. The roleosf these sites during protein synthesis are discussed in mored etail below.
Transfer RNA. Before the genetic code (q.v.) was understood, Francis Crick proposed the adaptor
hypothesis to explain how the nucleotide sequence in mRNA could be translated into protein. The
Protein Synthesis 31 5
model predicted the existence of an adaptor molecule which would recognize both the nucleic acid
sequence of the message and the appropriate aminoa cid, bringing thet wo together at the ribosome.
The adaptor molecule is transfer RNA (tRNA). The W A S are a relatively homogeneous family
of RNA molecules, usually 75-100 nucleotides in length, which are extensively processed during
their production (see RNA Processing). They possess a characteristic secondary and tertiary structure
(Box 23.2), most importantly the acceptor stem (to which the amino acid binds) and the anticodon
loop (which carries the three nucleotide anticodon that forms complementary base pairs
with codons in the mRNA). Bacterial cellcs ontain up to 35 different tRNA, ande ukaryotic cells up
to 50. This number is lower than the number of possible codons in the genetic code, but greater than
the number of amino acids specified by the code. This indicates that individual tRNAs can recognize
more than one codon (reflecting wobble pairing, q.v.), but that different tRNAs may be charged
with the same amino acids (these are isouccepting tRNA, q.v.).
The tRNAs are charged (conjugated to their corresponding amino acids) by enzymes termed
amino acyl tRNA synthetases. There is one enzyme for each amino acid, and therefore each
synthetase recognizes all its cognate isoaccepting tRNAs. The charging mechanism and its regulation
are considered in detail elsewhere (see The Genetic Code).
23.2 The mechanism of protein synthesis
Overview. The initiation stage of protein synthesis includes the assembly of the protein synthesis
components, the positioning of the ribosome at the translation start site, and the placement of the
first amino acid in the polypeptide (Figure 23.2). Initiation ends when the first peptide bond is
formed. In both prokaryotes and eukaryotes, the small ribosome subunit binds to the mRNA before
Jubduuu SD AUG

IFS, GDP 4
2&
m, GDP
4
Figure 23.1: Initiation of protein synthesis in bacteria and eukaryotes. In bacteria (left) the smalls ubunit
(1) binds to the Shine-Dalgarno (SD)se quence (2) and recruits the initiator tRNA (3),w hereupon the large
subunit binds (4). In eukaryotes (right), the small subunit (1) associates with elF-2-GTP and the initiator tRN IA
to form a preinitiation complex (2) which recognizes the cap-binding protein (CBP) at the 5' cap of the
mRNA, binds, and scans along until it finds an initiator codon in the correct context (3) before the large
subunit binds (4). For clarity, the small ribosome subunit is shown to span just six bases, although in reality it
spans 30-40 bases, allowing it to interact with the bacterial SD sequence (or the eukaryotic Kozak
sequence) and the initiation codon simultaneously. The A-site and P-site of the ribosome are indicated.
,.
31 6 Advanced Molecular Biology
the large subunit, and bindingr equires a number of initiation factors( IFS), which are released when
the large ribosome subunit is recruited. It is thus the small subunit that recognizes the ribosome
binding site on the mRNA. The first amino acid is attached to a special initiator tRNA, which has
the unique property of being able to enter the partial P-site on the small ribosome subunit. The
initiator codon is usually AUG; it marks the beginning of the open reading frame and sets the reading
frame for the rest of the polypeptide. All other elongator tRNAs enter the mmplete A-& in the
fully assembled ribosome during the elongation stage. Elongation involves a cycle of three reactions -
recruitment, transpeptidation and translocation. Each step requires elongation factors (EFs) and
the hydrolysis of GTP provides energy. Then et effecotf the three reactions is totr ansfer amino acids
from the cytoplasmon to the nascent polypeptide chain and to shunt the ribosome along the mRNA
in units of three nucleotide residues. Protein synthesis ceases when the ribosome encounters a termination
codon, where release factors attach to the ribosome and cause the translation
machinery to disperse.
hitiation in bacteria - detailed mechanism. In eubacteria, the 30s ribosomal subunit binds toth e
mRNA only when associated with initiation factor IF3, which stabilizes the structure of the small
subunit and prevents premature interaction with the large subunit. Although IF3 controls the ability
of the small subunit to bind to the mRNA, the specificity of the recognition is provided by the
ribosome itself. The ribosome binding site on the mRNA is the Shine-Dalgarno sequence (consensus
UAAGGAGG) and formsb ase pairs with a complementary sequencein the 16s rRNA of the
ribosome. The initiator codon (usually AUG but sometimes GUG or UUG) lies -10 nt downstream
of the Shine-Dalgarno sequence and correct positioning of the ribosome aligns the initiator codon
with the P-site of the ribosome subunit.
The initiator codon specifies the amino acid methionine. There are two tRNA carrying methionine
in E. coli. One (tRNAPt) specifically recognizes initiation codons and the other (tRNAmMet)
recognizes internal AUG codons (internal GUG and UUG codons are recognized by tRNAVa1 and
tRNALeU, respectively). Once the initiator tRNA has been charged, the methionine is modified by
formylation of the amino group. The signal for this may be unpairedr esidues in the terminal position
of the double-stranded acceptor stem, which are paired in elongator tRNAs (methionyltRNAmMect
annot be formylated). Thiss ignature also prevents the initiator tRNA entering the ribosomal
A-site during elongation, and the modification, since it blocks the amino group, prevents Nformylmethionine
formingp eptide bonds with upstreamr esidues. The initiator tRNA is recruited
to the P-site of the small ribosomes ubunit duringi nitiation. The Psite provides a special molecular
environment which allows access only to the initiator tRNA, and not to the elongator tRNAs. The
specificity of the initiator tRNA is conferred by three G:C base pairs in the anticodon stem, which
are absent from elongator WAS. N-formylation is not required for initiator specificity - initiator
tRNAs containing ordinary methionine are as efficient as correctly modified initiators. A degree of
wobble (q.v.) in the third anticodon position allows AUG, GUG and UUG to act as initiators, but
tRNAPt recognizes these codons with diminishinge fficacy; thus the choice of start codonc an be
used to establish a constitutive control over the efficiency of the initiation of protein synthesis.
The initiator tRNA is carried to the P-site by IF-2 in the presence of GTP. IF-l then binds to the
complete initiation complex to stabilize it. All initiation factors are released when the 50s subunit is
recruited. This process requires the hydrolysis of GTP to provide energy.
hitiation in eukaryotes - detailedmechanism. Eukaryotic and bacterial initiation mechanisms are
similar, the major differences reflectingth e order of assembly of the components, the larger number
of eukaryotic initiation factors, and that normal methionine is used both for initiation and elongation
in eukaryotes. Thus the initiator and elongator methionyl-tRNAs (tRNAimet and tRNAmmet,
respectively) carry identical amino acids and differ only in the structure of the tRNA itself.
In eukaryotes, the small ribosome subunit is unable to bind to the mRNA without first becoming
associated with the initiator tRNA. A ternary complex of tRNAimet, eIF-2 and GTP bind the small
Protein Synthesis 317
subunit to form the preinitiation complex, which then associates with the mRNA. The ribosome
binding site is defined by the initiation codon, although the sequence context surrounding this may
be important and is known as the Kozak consensus (ACCAUGG). Initially, the preinitiation complex
binds at the 5' 7meG cap structure and scans along the mRNA to find the first available initiation
codon. Several initiation factors are required for this process, including a cap binding protein
(eIF-4F), eIF-3, eIF-4A and eIF-4B. The cap binding protein facilitates initial binding of the 40s subunit.
eIF-4A and 4B cooperate to remove secondary structure from the 5' untranslated region (UTR),
then these and eIF-3 assist 40s subunit bindingt o form the initiation complex. The larges ubunit is
associated with a further initiation factor, eIF-6. Binding of the large subunit to the initiation complex
involves the loss of eIF-2 and eIF-3 from the preinitiation complex and the loss of eIF-6 from the
large subunit, a processm ediated bye IF-5. Initiation factors eIF-2 and eIF-6 are both antiassociation
factors: they prevent ribosome subunits interacting in the cytoplasm. The other initiation factors disassociate
from the ribosome as the large subunit binds, a process requiring GTP hydrolysis.
The elongation cycle. The elongation cycle, essentially identical in bacteria and eukaryotes, occurs
in three stages - recruitment, transpeptidation and translocation (Figure 23.2).
Following initiation, the initiator aminoacyl-tRNA occupies the ribosomal P-site and the next
codon is aligned with the A-site. The first elongator aminoacyl-tRNA enters the A site and its
anticodon pairs with the codon, a recognitionm ediated by ane longation factor (EF-Tu in bacteria,
eEF-1 in eukaryotes, each associated with GTP).
The peptide bond joining the initiator tRNA in the P-site to methionine is then broken and a new
peptide bond is formed between methionine anthde amino acid occupying the A-site. The initiator
tRNA is discharged. This transpeptidation reaction is mediated by the peptidyltransferase activity
of the large ribosomal subunit. The GTP associated with EF-Tu (or eEF-1) is hydrolyzed and the
elongation factor is ejected. EF-Tu/GDP is inactive, and the nucleotide is displaced by a second
elongation factor EF-Ts, which is itself displaced by GTP. EF-Tu can now be reused. There appears
to be no counterpart to EF-Ts in eukaryotes, although eEF-1 is an aggregate of several polypeptides,
one of which may perform the function of EF-Ts.
+GDP
Figure 23.2 The elongation cycle of protein synthesis. (1) Recruitment: a charged tRNA enters the ribosomal
A-site. (2) Transpeptidation: the peptide bond joining the amino acyl residue to the P-site tRNA is transferred
to the amino acyl residue occupying the A-site(.3 )T ranslocation: the spentt RNA is ejected from the P site
and the ribosome translocates three bases downstreamon the mRNA, shifting the A-site tRNA to the P-site
and aligning the next codon with the A-site, ready for recruitment of the next charged tRNA. The A-site and
P-site of the ribosome are indicated.
318 Advanced Molecular Biology
.1...1...1. 11111111111111111I UUUGCAUGGUAG
Figure 23.3 Termination of protein synthesis. When the ribosome encounters a termination codon, a release
factor binds in the A-site. This releases the nascent polypeptide from the peptidyl-tRNA in the P-site and
disassembles the ribosome, allowing the components of protein synthesis to be recycled.
The next stage is translocation. The initiator tRNA occupying the P-site is ejected (in bacteria it
is moved to a further ribosomal domain termed the E-site, whereas in eukaryotes the tRNA dissociates
from the ribosome). The methionyl residue is thus tethered to the amino acid occupying the
A-site by a peptide bond, and thtRe NA in the A-site is now referredt o as the peptidyl-tRNA. The
ribosome then translocates three bases along the mRNA while the peptidyl-tRNA remains associated
with its codon. The peptidyl-tRNA is thus transferred to the P-site and the mRNA moves
through the ribosome so that the next codon is aligned with the A-site. The geometry of the translocation
step depends upon thme RNA-tRNA interaction: mutant tRNAs with four base anticodons
cause four base translocations, resulting inframeshifting orfiumeshif suppression (q.v.). Translocation
requires a second elongation factor (EF-G in bacteria, eEF-2 in eukaryotes, also associated with GTP)
and involves GTP hydrolysis. In E. coli and some other bacteria, translocation also requires a free
rRNA species, 4.5s rRNA, whose role is unclear.
Termination of pmtein synthesis. Termination occurs when the ribosome encounters one of three
termination codons (q.v.). These usually have no cognate tRNAs, and translation ceases (however, c.f.
selenocysteine insertion, nonsense suppressors).T he completed polypeptide is released and the tRNA,
mRNA and ribosomal subunits disperse and are recycled forf urther translation events (Figure 23.3).
Termination requires a number of protein release factors (RFs) which bind in the A-site and recognize
the nonsense codons, These facilitate the release of the polypeptide from the last
peptidyl-tRNA, the ejection of the discharged tRNA from the ribosome, and the disassembly of the
ribosome. In E. coli there are two release factors recognizing different pairs of termination codons:
RF1 recognizes UAA and UAG, whereas RF2 recognizes UAA and UGA. Each is assisted by a third
factor, RF3. In eukaryotes, a single GTP-dependent factor (em) recognizes all three termination
codons, but not with the same efficiency. Readthrough may therefore occur, especially at weak
termini, reflecting competition between release factors and tRNA. The misreading of termination
codons is an important aspect of translational regulation (discussed below) and may be influenced
by secondary structure in the mRNA.
23.3 The regulation of protein synthesis
Constitutive contml of protein synthesis. The efficiency of protein synthesis is under constitutive
control, but may also be regulated either globally or at the level of individual transcripts.
Protein Synthesis 319
Constitutive levels of protein synthesis are dependent umpoRnN A structure and reflect such factors
as mRNA stability, the sequence and context of the ribosome binding site, the presence of secondary
structure, thec hoice of initiation codon, and codon bias throughout the open readifnrga me.
Secondary structure and the choice of termination codon also permit a group of regulatory
phenomena collectively described as programmed misreading, where the normal interpretatoifo n
the sequence of codons is suppressed. Examples of programmed misreading include readthrough,
selenocysteine insertion, and frameshifting. Readthrough occurs at weak termination codons (i.e.
termination codons where release factors are limiting), and there is competition between release
factors and tRNAs with tolerable anticodon sequences. Selenocysteine insertion occurs at the termination
codon UGA, and requires a secondary structure, the selenocysfeine insertion sequence (q.v.).
Frameshifting or recodinogc curs when ther ibosome pauses at a secondary structure or rcaored on,
shifts forwards or backwarbdys a single nucleotide, and continues translatioinn a different reading
frame. This can be used to change the reading frame midway through translation( e.g. in the retroviral
pol gene) to induce early truncation (e.g. the MS2 lysis gene) or to prevent truncation and
extend a gene product (e.g. in the E. coli dnaX gene).
In bacteria, the operon environment allows a novel form of translational regulation where the
translation of downstream genes is dependent on the translation of upstream genes in a polycistronic
message. Thes pacing of the individualo pen reading frames is important.W here the space
between successive open reading frames is greater than approximately 30 nucleotides, translation
begins with discrete initiation events and each locus is independent. If there is a shorter gap, the
ribosome can reinitiate by bridging the open readinfgra mes, i.e. without first dissociating from the
template. The initiation signal is different from the normal SDsequence, and spontaneouass sembly.
of ribosome subunits in the typical manner occurs with low efficiency. A mutation which blocks or
interrupts translation of the upstream gene therefore also effects translation of the downstream
gene; this is termed a polar mutation (e.g. q.v. lac operon).
The translation of eukaryotic mRNA is usually cap-dependent andf acilitated by scanningfo r the
first initiator codon. The picornavirus family provides an exception, in that the genomes contain
internal ribosome entry site(sI RES), i.e. motifs which form secondary structuresa llowing internal
initiation of protein synthesis. Internal initiation is not dependent upon the cap-binding proetIeFi-n
4F, and occurs when this protein is inactive (the picornaviridae block host protein synthesis by
inhibiting eIF-4F as parto f their infection strategy). However, no viral proteins arereq uired for IRES
function, i.e. initiation is dependent upon other host initiation factors. Internal initiation may also
occur for certain cellular transcripts, including Drosophila Anfennapedia and mouse fsT.he abundance
of IRES motifs and their significance in the control of gene expression is unknown; they have
been defined in functional terms and appear to share no conserved structural features.
Gl~barle gulation of protein synthesis.T he components of the 'basal apparatus' of protein synthesis
(thei nitiation, elongation and termination factors, and the components othf e ribosome) may be
regulated and may exercise a global control over protein synthesis. For example, several eukaryotic
viruses shut down host protein synthesis by phosphorylating the initiation factor eIF-2, and this
strategy may also be used by the cell itself where global repression of protein synthesis is required
(e.g. when cells are subjected to heat shock). Phosphorylation of eIF-2 in S. cerevisiae allows leaky
scanning, where the ribosome can skip weak initiators and bind to strongo nes. This lifts translational
repression of genes such as GCN4, where there are several unproductive AUG codons
between the cap and thdee finitive start codon on the mRNA. When eIF-2 is unphosphorylated, the
ribosome attempts to initiatep rotein synthesis at thef irst AUG, which is followed by an in-frame
termination codon.
Narrow domain regulation of protein synthesis. The translation of specific mRNAs can also be
regulated individually. Well-characterized examples include the mammalian ferritin mRNA and
ribosomal protein W mRNA in E. coli.
320 Advanced Molecular Biology
In the ferritins ystem, translation of ferritinm RNA is blocked when the concentration of intracellular
iron is low. The inhibitiodne pends upon iron-response elements( I R E S ) in the transcript,
and is mediatbeyd IRE-bindingp rotein, which is inactivateind t he presence of iron. The ferrIitRinE
is present in the 5 untranslated region of the transcript and IRE-BP binding inhibits protein
synthesis by preventing ribosomes canning. In the ferritinm RNA, IREs are not associated with AUrich
mRNA instability sites and do naoftf ect mRNA turnover, as is the case for transferrin receptor
mRNA (see RNA Processing).
The W system represents a feedback control for the synthesis of ribosomal components.
Ribosomal proteinS 8 binds toa stem loop structure formebdy residues 588-651 of the1 6s rRNA.
A similar structure is formbeyd t he 5 untranslated region and the 3f0ir bsat ses of the coding region
of the W ribosomal proteinm RNA. Protein S8 binds toW mRNA (albeit with lower efficiency than
does 165 rRNA) and inhibits protein synthesis. Excess 16s rRNA sequesters all the S8 protein and
thus derepresses W translation, whereas if 16s rRNA is limiting (and there isth erefore n o need for
W translation), the excess S8 protein preventsW translation.
A final example of specific translational regulation is providbedy antisense RNA (q.v.). This is found
mainly in prokaryote systems (e.g. in the control ofQ protein synthesis in bacteriophage h, in the control
of plasmid replicationg enes, and in the control oft ransposase synthesis in various transposons,
see Viruses, Plasmids, Mobile Genetic Elements), but also in eukaryotes (e.g. in the control oFf gf-2 synthesis
in the chick limb bud). More recently, antisense suppression of translation has been used as a
strategy to blockg ene function without in vitro or targeted mutagenesis allowing the effects of null phenotypes
to be determined withgoeunet disruption (q.v. transgenic organisms, gene therapy, cosuppression).