(iii)Deletion analysis. Sub-regions of the DNA template are excised and the effects of this deletion
on transcriptional initiation are observed either in an in vitroreaction or in living cells.
(iv) RNA start site mapping. The location of the 5-end of the RNA transcript is determined on its
DNA template.
(v) Footprinting. The binding site of RNA polymerase on the DNA template is determined.6.6.2.2.3 Promoter Structure in E. coli. Hundreds of E. colipromoters have been studied. The
majority of these show some sequence similarities, especially short conserved stretches. The strongest
consensus is the Pribnow box, a 6 bp sequence similar to the consensus TATAAT, but very few promoters
contain this exact sequence. The percentage chances of finding these bases in any given Pribnow box are:
T80 A95 T45 A60 A50 T96. The other conserved sequence found in many E. colipromoters, the35 box,
has the following consensus: T82 T84 G78 A65 C54 A45.
Both the35 and Pribnow boxes are very sensitive to sequence change. In general, mutations that make
the sequence less like the consensus tend to weaken the promoter and vice versa. The strongest promoter
is a combination of the two consensus boxes. It is important to remember that not all promoters need to be
strong. Different genes need to be transcribed at different rates in an organism. In keeping with the obser-
vations from in vitro transcription studies (Figure 6.17) mutations in the 35 box alter the rate of closed
complex formation, not the conversion into open complex, whereas mutations in the Pribnow box do not
affect closed complex formation and have the opposite effect.
Genes and Genomes 225
Figure 6.17 Initiation of transcription in E. coli. Sigma (s) factor. The RNA transcript is shown in red