that other regulatory sequences outside the coding region,
such as the 3^0 and 5^0 UTR, may influence translation efficiency
as well and can also be inserted into the reporter construct,
either on their own or together with the coding sequence. If
the goal of the experiment is not related to the regulation of a
specific gene, but rather to study global translational control
mechanisms, the specific mRNA sequence inserted down-
stream of the SunTag sequence may not be critical and different
sequences can be inserted. Thelengthof the reporter sequenceFig. 4Interpreting GFP fluorescence intensities of translation sites. The scFv-
GFP intensity associated with a single ribosome depends on the location of the
ribosome along the mRNA. The GFP intensity will initially increase as the
ribosome synthesizes successive SunTag peptides (illustrated in the figure by
the binding of 1, 2, or 3 scFv-GFP antibodies). Ribosome-associated fluores-
cence reaches a maximum once all SunTag peptides have been synthesized and
will remain constant while the ribosomes translate the remaining sequence of
the mRNA. As a consequence, ribosomes at the 3^0 end of the mRNA are labeled
brighter, than those at the 5^0 end. When the number of ribosomes on an mRNA is
calculated based on GFP intensity, these position-dependent effects of GFP
intensity need to be taken into account. A simple mathematical model can be
used, as described in the text
Imaging Translation in Live Cells 397