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Insights provided by ribosome profiling With these advantages and disadvantages in mind, the application of ribosome profiling to specific biologi- cal questions has confirmed much of what we know about translation mechanism from decades of elegant structural, biochemical and genetic studies50.Quantitative measurement of protein synthesis rates over multiple time points of a dynamic process can also provide information about specific gene function.The application of this method to numerous organisms and cellular states has illuminated fundamental aspects of cell biology that were previously challenging to probe experimentally, providing measurements for how much of each protein is synthesized, how translation is regu- lated, where synthesis starts and stops and what is being synthesized.Assuming that the average translation elongation rate is similar for different genes, ribosome profiling provides direct, global and quantita- tive measurements of rates of protein synthesis, thereby capturing information that has been largely invisible to gene expression measurements of mRNA levels alone.Analysis of the positions of mRNAs in polysome gradients provides valuable complementary information to that obtained with ribosome profiling, but again, this method is labo- rious and typically yields only a qualitative measure of protein synthesis.Other recent studies in disparate systems -- from the Drosophila melanogaster oocyte-to-embryo transi- tion55 to the Trypanosome life cycle56 to the mamma- lian cell cycle57 to plants under hypoxic conditions27 -- have used ribosome profiling to identify specific proteins that drive these complex processes.The latter category includes cellular differentiation, organismal develop- ment and dynamic responses to cellular stress, which are all cases in which the instantaneous and downstream gene expression measurements provided by ribosome profiling are particularly illuminating for understanding molecular control.Cases in which ribosome profiling data provide markedly dif- ferent information than can be obtained by traditional mRNA abundance measurements for gene expression tend to fall into two categories: systems in which tran- scriptional regulation is minimal26,54,55; and dynamic cellular programmes11,12,27,35,57-59.This property of proportional synthesis, by which sub- units of multiprotein complexes are synthesized at rates that are proportional to their stoichiometry in the com- plex, turns out to be generally true for Escherichia coli and was also observed for some (but not all) complexes in budding yeast (Saccharomyces cerevisiae).Subsequent morpholino knockdown experiments showed that specifically blocking translation of these three factors resulted in a shutdown of the first wave of zygotic tran- scription and development, indicating that they are the key factors responsible for the initiation of the zygotic developmental programme54.As is the case for many protein complexes in bacteria, the eight different subunits of the Fo F1 -ATP syn- thase are expressed from a single polycistronic mRNA, and thus measurements of mRNA levels would suggest that the subunits are all expressed at very similar levels.For example, hierarchical clustering of patterns of new pro- tein synthesis for each gene over the dynamic process of meiosis in budding yeast resulted in an intricate map of gene expression that provided highly detailed functionalHow much?3b).

Insights provided by ribosome profiling
With these advantages and disadvantages in mind, the
application of ribosome profiling to specific biologi-
cal questions has confirmed much of what we know
about translation mechanism from decades of elegant
structural, biochemical and genetic studies50. Ribosome
profiling has also made it possible to monitor transla-
tion with unprecedented depth and precision, providing
important — and at times surprising — insights. The
application of this method to numerous organisms and
cellular states has illuminated fundamental aspects of
cell biology that were previously challenging to probe
experimentally, providing measurements for how much
of each protein is synthesized, how translation is regu-
lated, where synthesis starts and stops and what is being
synthesized.
How much? A quantitative view of protein synthesis. The
simplest and broadest application of ribosome profiling
is as a quantitative proteomics tool to monitor which
proteins are being synthesized, and at what levels, thus
providing rich molecular insight into a given cell state.
Ribosome footprint density reflects the number of ribo-
somes at a given position. Assuming that the average
translation elongation rate is similar for different genes,
ribosome profiling provides direct, global and quantita-
tive measurements of rates of protein synthesis, thereby
capturing information that has been largely invisible to
gene expression measurements of mRNA levels alone.
Mass spectrometry can, in principle, be used to measure
rates of protein synthesis; however, this is technically
difficult, as it typically requires metabolic labelling and
multiple measurements per sample. Analysis of the
positions of mRNAs in polysome gradients provides
valuable complementary information to that obtained
with ribosome profiling, but again, this method is labo-
rious and typically yields only a qualitative measure of
protein synthesis.
In many cases, the ability to observe new protein syn-
thesis globally and quantitatively provides insights that
are not apparent from measurements of mRNA abun-
dance. Bacterial operons provide a vivid example of the
value of being able to directly measure rates of protein
synthesis. As is the case for many protein complexes in
bacteria, the eight different subunits of the Fo
F1
-ATP syn-
thase are expressed from a single polycistronic mRNA,
and thus measurements of mRNA levels would suggest
that the subunits are all expressed at very similar levels.
Ribosomal profiling, however, shows that the individual
ORFs that encode the subunits of the Fo
F1
-ATP synthaseinformation12. In these data, the genes responsible for
the complex, conserved and meiosis-specific processes
of homologous recombination and synaptonemal com-
plex assembly emerged as a single cluster of 46 genes.
This observation was surprising, because these pro-
cesses are known to be regulated extensively at the post-
translational level, and also because the cluster included
almost every gene that had been found through decades
of intensive genetic and cytological screening focused
on these processes. This cluster also included several
uncharacterized genes, two of which (GMC1 and GMC2)
were subsequently shown to have roles in recombination
and synaptonemal complex formation12,53.
Another striking recent example of this type of
analysis used ribosome profiling to identify the factors
that are responsible for initiation of the zygotic develop-
mental programme in zebrafish54 (FIG. 3b). The initiation
of zygotic development in vertebrates depends heavily
on translational control, as maternal mRNAs provide
the starting pool of material for translation. Zygotic
activation then requires destruction of these mater-
nal mRNAs and transfer of developmental control to
the zygote itself. To determine the factors that medi-
ate the first wave of zygotic transcription, ribosome
profiling data were analysed for samples collected just
before zygotic activation. This study identified Nanog,
Sox19b and Pou5f1 as the three transcription factors
that were most heavily translated, from the large pool
of maternal mRNAs at this stage (FIG. 3b). Subsequent
morpholino knockdown experiments showed that
specifically blocking translation of these three factors
resulted in a shutdown of the first wave of zygotic tran-
scription and development, indicating that they are the
key factors responsible for the initiation of the zygotic
developmental programme54.
Other recent studies in disparate systems — from
the Drosophila melanogaster oocyte-to-embryo transi-
tion55 to the Trypanosome life cycle56 to the mamma-
lian cell cycle57 to plants under hypoxic conditions27
— have used ribosome profiling to identify specific
proteins that drive these complex processes. Cases in
which ribosome profiling data provide markedly dif-
ferent information than can be obtained by traditional
mRNA abundance measurements for gene expression
tend to fall into two categories: systems in which tran-
scriptional regulation is minimal26,54,55; and dynamic
cellular programmes11,12,27,35,57–59. The latter category
includes cellular differentiation, organismal develop-
ment and dynamic responses to cellular stress, which
are all cases in which the instantaneous and downstream
gene expression measurements provided by ribosome
profiling are particularly illuminating for understanding
molecular control.
How? Insights into the mechanism of translational
control. The basic mechanism by which the riboso-
mal machinery reads codon information in mRNAs to
create proteins is conserved, and many features of this
process are well understood50. Nonetheless, there are
aspects of translational control that are not amenable
to recapitulation in vitro and for which results fromoperon are translated at a ratio of 1:1:1:1:2:3:3:10.
Remarkably, these ratios precisely reflect the stoichiome-
try of these components in the ATP synthase51,52 (FIG. 3a).
This property of proportional synthesis, by which sub-
units of multiprotein complexes are synthesized at rates
that are proportional to their stoichiometry in the com-
plex, turns out to be generally true for Escherichia coli
and was also observed for some (but not all) complexes
in budding yeast (Saccharomyces cerevisiae). Such meas-
urements of instantaneous rates of protein synthesis may
prove to be a general tool for exploring how proteins
assemble and function together51.
Quantitative measurement of protein synthesis rates
over multiple time points of a dynamic process can also
provide information about specific gene function. For
example, hierarchical clustering of patterns of new pro-
tein synthesis for each gene over the dynamic process of
meiosis in budding yeast resulted in an intricate map of
gene expression that provided highly detailed functional