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Transcription is the fundamental process by which RNA is synthesized by RNA polymerases on double-stranded DNA templates. Bacteriophage T7 RNA polymerase is a DNA-dependent RNA polymerase that is encoded in the T7 bacteriophage genome. The enzyme consists of a single polypeptide chain that has a molecular weight of 99 KDa. It catalyzes the processive polymerization of messenger RNA from nucleoside triphosphate precursors by using one strand of DNA as a template . Biochemical and biophysical studies have shown that the enzyme has an stringent specificity for its promoter, initiates the synthesis of RNA from a single nucleotide, abortively cycles the synthesis of short transcripts (2-8 nt), is regulated by transcriptional inhibition, and can terminate transcription (Figure 1). In this paper, I will present a general description of the most important aspects concerning T7 RNA polymerase. I will first look at the primary amino acid sequence and locate those amino acids that are involved in promoter recognition and catalysis. Based on recent biochemical and x-ray crystallographic studies, I will then examine in detail the structural characteristics of the enzyme. Special attention will be paid to those structural motifs that are responsible for DNA promoter recognition and RNA synthesis.
I will then examine the catalytic mechanism of the T7 RNA polymerase with an emphasis of the different stages. Based on recent studies, I will provide evidences supporting the transition from transcription initiation to elongation and to the abortive intermediate transcripts. I will then show the kinetic parameters corresponding for transcription initiation, and then I will describe the two-metal ions catalytic mechanism of the enzymes. Finally, I will briefly go over the evidences that support a common ancestor for the nucleic acid polymerases. I will compare important structural motifs that are present in the Klenow fragment, HIV-1 RT, and T7 RNA polymerase. And lastly, I will consider the factors that are involved in the unique specificity found in some polymerases.
Genetic Analysis of T7 RNA Polymerase: The genome of bacteriophage T7 was sequenced by Dunn et. al. 1983 and Moffatt et. al., 1984. The whole DNA sequence consists of 399,336 base pairs. The coding region of T7 RNA polymerase is located between nucleotides 3170 and 5819. Thus T7 RNA polymerase consists of 883 amino acid residues (Figure 2 and Figure 3).
1 MNTINIAKND FSDIELAAIP FNTLADHYGE RLAREQLALE HESYEMGEAR 51 FRKMFERQLK AGEVADNAAA KPLITTLLPK MIARINDWFE EVKAKRGKRP 101 TAFQFLQEIK PEAVAYITIK TTLACLTSAD NTTVQAVASA IGRAIEDEAR 151 FGRIRDLEAK HFKKNVEEQL NKRVGHVYKK AFMQVVEADM LSKGLLGGEA 201 WSSWHKEDSI HVGVRCIEML IESTGMVSLH RQNAGVVGQD SETIELAPEY 251 AEAIATRAGA LAGISPMFQP CVVPPKPWTG ITGGGYWANG RRPLALVRTH 301 SKKALMRYED VYMPEVYKAI NIAQNTAWKI NKKVLAVANV ITKWKHCPVE 351 DIPAIEREEL PMKPEDIDMN PEALTAWKRA AAAVYRKDRA RKSRRISLEF 401 MLEQANKFAN HKAIWFPYNM DWRGRVYAVS MFNPQGNDMT KGLLTLAKGK 451 PIGKEGYYWL KIHGANCAGV DKVPFPERIK FIEENHENIM ACAKSPLENT 501 WWAEQDSPFC FLAFCFEYAG VQHHGLSYNC SLPLAFDGSC SGIQHFSAML 551 RDEVGGRAVN LLPSETVQDI YGIVAKKVNE ILQADAINGT DNEVVTVTDE 601 NTGEISEKVK LGTKALAGQW LAHGVTRSVT KRSVMTLAYG SKEFGFRQQV 651 LEDTIQPAID SGKGPMFTQP NQAAGYMAKL IWESVSVTVV AAVEAMNWLK 701 SAAKLLAAEV KDKKTGEILR KRCAVHWVTP DGFPVWQEYK KPIQTRLNLM 751 FLGQFRLQPT INTNKDSEID AHKQESGIAP NFVHSQDGSH LRKTVVWAHE 801 KYGIESFALI HDSFGTIPAD AANLFKAVRE TMVDTYESCD VLADFYDQFA
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