Nucleic Acids Research, 2008, Vol. 36, No. 16 5265 Figure 3. Initiating nucleotide in primers synthesize by A. aeolicus primase on the preferred d(CCC) template. (A) Chromatograms of primers produced as a function of CTP concentration and in the absence of ATP, GTP, or UTP, but with the presence of 400 mM CTP as denoted. (B) The fraction of synthesized RNA polymers 14 nt and longer in the presence of various CTP concentrations was quantiﬁed and then ﬁt to a modiﬁed Michaelis–Menten equation that allowed for activity in the absence of CTP. The ﬁt indicated that Y(0 mM CTP) = 27 1%, K50% = 16 1 mM and R2 = 0.994. The error bars represent the standard deviation of triplicate experiments and in most cases the error was smaller than the symbol on the plot. primer synthesis terminating prior to reaching the end of the template. Identification of initial ribonucleotide in primer synthesized by A. aeolicus primase The impact on de novo primer synthesis by A. aeolicus primase in the absence of GTP, UTP, ATP and CTP was evaluated to further verify trinucleotide speciﬁcity and to identify the initial ribonucleotide copied into the primer. No primers were synthesized when GTP or UTP were omitted from the reaction with the preferred d(CCC) template (Figure 3A), consistent with the require- ment for two GTPs during dinucleotide formation or initiation and the requirement for GTP and UTP during elongation. The omission of ATP from reactions with either the d(CCC) template (Figure 3A) or d(GCC) tem- plate (data not shown) increased A. aeolicus primase activ- ity by 10%. This was expected since both templates lacked thymine and the absence of ATP most likely enhanced the activity of A. aeolicus primase by reducing the time needed to sample the NTP pool at each template position during primer synthesis (36). In the absence of CTP with the d(GCC) template, no primers were generated, indicating that A. aeolicus primase required this ribonu- cleotide for dinucleotide polymerization (data not shown). However, increases in the concentration of CTP resulted in a proportional increase in the amount of full- length 17-mer primers, suggesting that A. aeolicus primase initiated primer synthesis complementary to the middle cytosine in the recognition trinucleotide d(GCC). To verify that the 17-mer RNA primers synthesized by A. aeolicus primase initiated complementary to the middle nucleotide in its recognition sequences, CTP titration experiments were performed with the preferred d(CCC) template. The template was designed so that the omission of CTP generated a primer that was 3 nt shorter than full-length RNA primers. When the preferred d(CCC) template with the unique guanine at the antepenultimate position was used in the CTP titration experiments, the absence of CTP in the primer synthesis reactions resulted in predominately a 14-mer RNA polymer (Figure 3A). These data conﬁrmed that the initial ribonucleotide in the primers produced by A. aeolicus primase was com- plementary to the middle nucleotide in the recognition trinucleotide. Interestingly, in the presence of CTP and the other NTPs, a minor peak adjacent to the predominant full- length 17-mer was apparent (Figures 2 and 3). Since the elution time for each RNA polymer length is spaced by about 0.3 min, the minor peak that eluted just prior to each 17-mer was due to a diﬀerence in sequence composi- tion and not length, indicating misincorporation. This observation was further supported when CTP was omitted from the primer synthesis reactions with the preferred d(CCC) template. In the absence of CTP with the pre- ferred d(CCC) template containing a unique guanine at the antepenultimate position, some primers were longer than the expected 14-mer, indicating that A. aeolicus pri- mase was misinserting nucleotides (Figure 3). In fact, the ﬁt to the data indicated that 27 1% of the primers were longer than expected, giving a misinsertion rate similar to the primases from S. aureus and E. coli (31,34). As the CTP concentration was increased, the percent of primers that were longer than the 14-mer increased in a saturable manner to give a K50% of 16 1 mM CTP during the elongation part of the reaction (Figure 3B). This KM- like kinetic aﬃnity during the elongation step was similar to the same parameter for primases from S. aureus and