Function Analysis Of Initiator Element In Sulfolobus Promoter

It is well know that Initiator (Inr) is an important basal promoter element for transcription in eukaryotes. Considerable efforts have been applied toward the properties of eukaryotic Inr, it is a unidirectional core promoter element overlapping the transcription start site(TSS). TATA-box and Inr elements strongly synergized with each other to enhance transcription when both were present in the same promoter, whereas, Inr can determine the direction of transcription and start site localization without other core promoter elements.However, archaea research have a late start, genetic systems and genetic manipulation is not available until recently, the archaeal Inr (alnr) has been considered less important, since transcription is initiated efficiently in promoters with insertion or deletions between the TATA box and the Inr. Now we only known mutations in TSS region will reduce promoter strength and impact transcription start site selection, but the specific properties of alnr is not clear including the location, the length, sequence preferences and conservative of alnr.It is becoming clear that well-characterized mechanisms in bacteria and archaea have functional counterparts in higher organisms, so the bacterial and archaea systems offer us an advantage in thinking about possible mechanisms utilized by eukaryotes. Kimberly B. Decker and Deborah M. Hinton found RNA-synthesizing cores of multisubunit polymerases are structurally and functionally similar in bacteria, archaea and eukaryotic, promoter modularity is conserved from bacteria to humans, bacteria, archaea and eukaryotes utilize similar regulation strategies close to or within the core promoter: different combinations of core promoter elements. Inr is a paradigm for core promoter heterogeneity contribute to combinatorial gene regulation in eukaryotes. This study in alnr can improve the understanding not only about archaea promoter structure and transcription initiation mechanism but also about transcription regulation in core promoter level.In this study, Inr mutants were constructed by overlapping PCR and reverse PCR. S. islandicus reporter gene system was employed in this study. In S. islandicus reporter gene system, lacS gene is used as the reporter gene, pZCl as E. coli-Sulfolobus shuttle vector and S. islandicus E233S ApyrEF/AlacS as host. After transformation of reporter plasmids into S. islandicus E233S, transformants were obtained and corresponding lacS activities were detected to evaluated the effect of expression, RT-qPCR and primer extension were used to determine the impact of mutations on the transcription, thus the impact on translation can be calculated according to the lacS activity. On the other hand, bioinformatic analysis were used to investigate conserved Inr sequence of S. solfataricus P2, so we can combined molecular genetics analysis with bioinformatic methods to analyse alnr.the dramatic effect of+1to+6mutations on araS promoter support the presence of araS Inr, primer extension and RT-qPCR confirmed that this elements played an important role in transcription but had a limited effect on translation. Inr of T6BRE was mutanted and lacS activity dropped to2.1%of D-55,comfirmed that Inr is not only function in araS promoter but also contributes to basal promoter activity. The promoter activity of mutant TAmut was completely abolished due to a transversion mutation in the TATA-box of the constitutive hybrid promoter T6BRE1, this indicated that the Sulfolobus Inr was incapable of initiating transcription independently of the TATA-box. Based on the results of nucleotide preference identification analysis, the wild-type sequence of the Inr in the araS promoter was found to be the most functional, a preferential sequence of the functional Inr in the araS promoter can be summarize as+1GAKAMY+6(IUPAC, K=G or T; M=A or C; Y=T or C).The conserved motif derived from S. solfataricus P2was-1YRWKAAA+6, which was almost identical to the functional araS Inr sequence. We divide the promoters into2groups according to their5’UTR, the results indicated that promoters of genes containing a (>4nt)5’UTR tend to harbor Inr. Then the promoters were divided into five groups according to their different ruction:Inr is conserved in energy metabolism genes rather than other four subgroups. Obviously not all genes contain Inr, experimental results on SiReO562, SiRe2130, SiRe2129and SiRe2125also confirm that, they regulated by arabinose like araS but not harbor Inr. Remarkable, Inr mutations decrease expression significantly whether in araS promoter or promoters of randomly chosen Sso1934, Sso3180and Sso1171.Adding the araS Inr to the3’end of the SiRe0562, sso0009and sso1029promoters, which are Inr-less genes, the expression of reporter gene has been enhanced. This suggested that the presence or absence of an Inr might determine the promoter activities and globally regulate gene expression in Sulfolobus, which may depends on the binding of TFB and RNA polymerase to Inr. The mechanism of Inr-involved global gene regulation is, somehow, similar to that of family modulated transcription in bacteria and GTF combination-involved global regulation in halophiles. Sulfolobus encodes only one TBP and two functional TFBs, which is not sufficient for transcriptional regulation using the mechanism mentioned. This suggests that the Inr may compensate for the limited number of GTFs in Sulfolobus for global gene regulation by binding to TFB or KNAP subunits with different affinities.Overall, this work combine in vivo experiment and bioinformatic analysis to study alnr, we do indentified a conserved aInr and confirmed that this elements played an important role in transcription. Inr is conserved in energy metabolism genes and promoters of genes containing a (≥4nt)5’UTR tend to harbor Inr. The araS Inr enhances the strength of Inr-less promoters in Sulfolobus. To our knowledge, those results are the first experimental results that confirmed the functioning of Inr in archaeal.