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Category: Microbial Genetics and Molecular Biology
Recent Studies of RNase P † , Page 1 of 2
< Previous page | Next page > /docserver/preview/fulltext/10.1128/9781555818333/9781555810733_Chap06-1.gif /docserver/preview/fulltext/10.1128/9781555818333/9781555810733_Chap06-2.gifAbstract:
This chapter is primarily a progress report on work with the enzyme from Escherichia coli. RNase P, the endonuclease responsible for the biosynthesis of the 5' termini of mature tRNA, is a ribonucleoprotein. The chapter primarily focuses on relationships between the structure and function of the subunits of RNase P, as determined from studies with the enzyme from eubacterial sources. Two important goals of current research on RNase P are identification of the active site of the enzyme and elucidation of the details of the chemical reaction governed by it. Several lines of evidence have implicated the region in M1 RNA that contains at least nucleotides 60 to 92, 230 to 260, and 290 to 360 as being essential for catalysis. The evidence comes from analysis of deletion mutants, studies of the binding of both individual tRNA precursors and the protein cofactor to M1 RNA, and studies of the binding of divalent metal ions to M1 RNA. Some point mutations that significantly affect the activity of M1 RNA are also located in these regions. A summary of these results is shown in the chapter. The protein cofactor of RNase P from E. coli (C5 protein) is a highly basic molecule of 119 amino acids, with a molecular mass of 13,800 daltons.
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Secondary structures of substrates for RNase P. (A) The precursor to tyrT(SuUAG) (pTyr) from E. coli. (В) The precursor to 4.5S RNA ( 9 ). (С) Model substrate derived from tRNAPhe: from E. coli ( 52 ) and one composed of two oligoribonucleotides, with the external guide sequence (EGS) indicated ( 56 ).
Secondary structures of substrates for RNase P. (A) The precursor to tyrT(SuUAG) (pTyr) from E. coli. (В) The precursor to 4.5S RNA ( 9 ). (С) Model substrate derived from tRNAPhe: from E. coli ( 52 ) and one composed of two oligoribonucleotides, with the external guide sequence (EGS) indicated ( 56 ).
Proposed secondary structure of RNA subunit (Ml RNA) of RNase Ρ from E. coli (A, left) and general model, based on phylogenetic comparisons, for all RNA subunits of RNase Ρ (В, right). In (A), nucleotides that can be deleted without major negative effects on catalytic function ( 27 ) are boxed. In (B), absolutely conserved nucleotides in RNAs from RNase Ρ from various bacteria are shown in capital letters; nucleotides that are not invariant, but are conserved in at least 90% of the available sequences, are shown in lower case letters. Nucleotides that are conserved in fewer than 90% of RNAs are shown with filled circles (•). Nucleotides that are not present in all sequences, but are absent from fewer than 10% of the available sequences, are indicated with open circles (°); nucleotides absent from more than 10% of the sequences are not shown. Reprinted in part from reference 15 with permission.
Proposed secondary structure of RNA subunit (Ml RNA) of RNase Ρ from E. coli (A, left) and general model, based on phylogenetic comparisons, for all RNA subunits of RNase Ρ (В, right). In (A), nucleotides that can be deleted without major negative effects on catalytic function ( 27 ) are boxed. In (B), absolutely conserved nucleotides in RNAs from RNase Ρ from various bacteria are shown in capital letters; nucleotides that are not invariant, but are conserved in at least 90% of the available sequences, are shown in lower case letters. Nucleotides that are conserved in fewer than 90% of RNAs are shown with filled circles (•). Nucleotides that are not present in all sequences, but are absent from fewer than 10% of the available sequences, are indicated with open circles (°); nucleotides absent from more than 10% of the sequences are not shown. Reprinted in part from reference 15 with permission.
Hypothetical cage structure for RNA subunits of RNase P, as illustrated by cage for RNA from RNase Ρ of В. subtilis ( 20 ). The vertical lines below the lowermost sequences indicate nucleotides that can form base pairs with sequences (not shown) in the central portions of the RNA. Nucleotides depicted in lowercase letters are conserved in terms of both nature and position in the RNAs from RNase Ρ from eubacterial sources. The structures shown in Fig. 2 are based on more recent phylogenetic comparisons and differ in a few details in helical regions from that shown in Fig. 3 .
Hypothetical cage structure for RNA subunits of RNase P, as illustrated by cage for RNA from RNase Ρ of В. subtilis ( 20 ). The vertical lines below the lowermost sequences indicate nucleotides that can form base pairs with sequences (not shown) in the central portions of the RNA. Nucleotides depicted in lowercase letters are conserved in terms of both nature and position in the RNAs from RNase Ρ from eubacterial sources. The structures shown in Fig. 2 are based on more recent phylogenetic comparisons and differ in a few details in helical regions from that shown in Fig. 3 .
Proposed secondary structure of RNA subunit of RNase Ρ from human tissue. Nucleotides (79–84 and 318–323) in bold letters participate in pseudo-knot formation ( 5a ).
Proposed secondary structure of RNA subunit of RNase Ρ from human tissue. Nucleotides (79–84 and 318–323) in bold letters participate in pseudo-knot formation ( 5a ).
Proposed pathway for the activation of substrates in the reaction catalyzed by RNase Ρ (57). P1 and P2 refer to the products of cleavage of S by RNase P.
Proposed pathway for the activation of substrates in the reaction catalyzed by RNase Ρ (57). P1 and P2 refer to the products of cleavage of S by RNase P.
Regions of tRNA precursor important in determination of site and rate of cleavage by RNase P. The ovals surround critical regions of the phosphodiester backbone. Conserved nucleotides in the P loop and the 3′ terminal CCA sequence, as well as the very common G · С pair at the base of the acceptor stem, are shown. The arrow indicates the site of cleavage of RNase P.
Regions of tRNA precursor important in determination of site and rate of cleavage by RNase P. The ovals surround critical regions of the phosphodiester backbone. Conserved nucleotides in the P loop and the 3′ terminal CCA sequence, as well as the very common G · С pair at the base of the acceptor stem, are shown. The arrow indicates the site of cleavage of RNase P.