Structural Analyses of the Ribosome by Chemical Modification Methods - Biophysical Approaches to Translational Control of Gene Expression - page 77

Every so often new mobility shifts have to be created as capillary decay will
cause minor changes in the shift speed.
4. Signal decay correction—hSHAPE works by detecting reverse transcriptase
stops caused by adducts formed by 1M7. Since adducts may form anywhere
downstream of a primer, this tends to favor the creation of shorter cDNA frag-
ments. Additionally, reverse transcriptase is imperfect and will not perfectly
extend infinitely. Thus, as the length of a trace increases, the signal height
decreases. Signal decay correction corrects for this by applying a statistical
model to correct peaks heights. After correction, intense peaks at the beginning,
middle, and end of a given selection should be approximately the same.
5. Scaling—Any of the previous steps can affect the scale of each of the primers
relative to one another. The Scaling option allows the user to correct trace heights
so that the lowest 5-10% of the experimental and control peaks are approxi-
mately the same. Once this correction is applied, peaks heights are quantitatively
equalized relative to each other.
6. Align and Integrate—This is the core of the ShapeFinder program. By this point,
all peaks are appropriately relative, peaks are aligned with equivalent peaks in
each channel, and baselines have been set to zero. After the region of interest is
chosen, the range of bases to which they correspond are entered and an appropri-
ate sequence file is selected. The program attempts to find all peaks, match the
equivalent peak in each channel, and align them to the chosen sequence. Next,
the user is able to manually remove or add peaks that were missed during the
automated selection and find corresponding peaks in the sequencing channels.
Once all peaks are matched and aligned to the appropriate sequence, the program
defines the area under each peak. A file is produced containing the area under
each curve, both experimental and control, their standard deviations, and the
absolute SHAPE reactivity of each position, defined by subtracting the control
area of a peak from the equivalent experimental area. This file also determines
the sequence identity of each peak. From this point further downstream analyses
may be performed by the user.

4.4.7 Completed hSHAPE Projects

Since its development, the hSHAPE protocol has been applied to numerous struc-
tural challenges. In 2009, the entire HIV-1 genome was analyzed in vitro (Watts
et al. 2009 ). This examination helped show how secondary and tertiary structure
elements of the HIV-1 genome are involved in numerous regulatory functions,
including the gag-pol frameshift signal. The reactivity and flexibility of the entire
salt-washed yeast ribosome was completed in 2011 (Leshin et al. 2011 ) . A complete
salt-washed structure map is the first step in better understanding the structure of
eukaryotic translation Fig. 4.2 .