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RNA PolymeraseIII Class I= 5S RNA ClassII = tRNA ClassIII = U6 snRNA Transcription Factors = TFIII TFIII A TFIII B= TBP+ BRF TFIII C Clover leaf L shape.

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Presentation on theme: "RNA PolymeraseIII Class I= 5S RNA ClassII = tRNA ClassIII = U6 snRNA Transcription Factors = TFIII TFIII A TFIII B= TBP+ BRF TFIII C Clover leaf L shape."— Presentation transcript:

1 RNA PolymeraseIII Class I= 5S RNA ClassII = tRNA ClassIII = U6 snRNA Transcription Factors = TFIII TFIII A TFIII B= TBP+ BRF TFIII C Clover leaf L shape Pre-tRNA D-loop Anticodon loop TψC loop RNase= Endonuclease Exonuclease RNase P---- 5’P (Endo) RNase F---- 3’ OH (Endo) RNase D---- 3’ OH (Exo)

2 Initiation Initiation: the construction of the polymerase complex on the promoter. Pol III is unusual (compared to Pol II) requiring no control sequences upstream of the gene, instead normally relying on internal control sequences - sequences within the transcribed section of the gene (although upstream sequences are occasionally seen, eg. U6 snRNA gene has an upstream TATA box as seen in Pol II Promoters).TATA box [edit] Class IIedit Typical stages in a tRNA (also termed class II) gene initiation:tRNA TFIIICTFIIIC (Transcription Factor for polymerase III C) binds to two intragenic (lying within the transcribed DNA sequence) control sequences, the A and B Blocks (also termed box A and box B).[1].DNA[1] TFIIIC acts as an assembly factor that positions TFIIIB to bind to DNA at a site centered approximately 26 base pairs upstream of the start site of transcription. TFIIIBTFIIIB (Transcription Factor for polymerase III B), consists of three subunits: TBP (TATA Binding Protein), the Pol II transcription factor TFIIB-related protein, Brf1 (or Brf2 for transcription of a subset of Pol III-transcribed genes in vertebrates) and Bdp1.[2].TBPBrf1Brf2[2] TFIIIB is the transcription factor that assembles Pol III at the start site of transcription. Once TFIIIB is bound to DNA, TFIIIC is no longer required. TFIIIB also plays an essential role in promoter opening. TFIIIB remains bound to DNA following initiation of transcription by Pol III (unlike bacterial σ factors and most of the basal transcription factors for Pol II transcription). This leads to a high rate of transcriptional reinitiation of Pol III-transcribed genes. [edit] Class Iedit Typical stages in 5S rRNA (also termed class I) gene initiation:5S rRNA TFIIIATFIIIA (Transcription Factor for polymerase III A) binds to the intragenic (lying within the transcribed DNA sequence) 5S rRNA control sequence, the C Block (also termed box C).DNA TFIIIA Serves as a platform that that replaces the A and B Blocks for positioning TFIIIC in an orientation with respect to the start site of transcription that is equivalent to what is observed for tRNA genes. Once TFIIIC is bound to the TFIIIA-DNA complex the assembly of TFIIIB proceeds as described for tRNA transcription. [edit] Class IIIedit Typical stages in a U6 snRNA (also termed class III) gene initiation (documented in vertebrates only):U6 snRNA SNAPcSNAPc (SNRNA Activating Protein complex) (also termed PBP and PTF) binds to the PSE (Proximal Sequence Element) centered approximately 55 base pairs upstream of the start site of transcription. This assembly is greatly stimulated by the Pol II transcription factors Oct1 and STAF that bind to an enhancer-like DSE (Distal Sequence Element) at least 200 base pairs upstream of the start site of transcription. These factors and promoter elements are shared between Pol II and Pol III transcription of snRNA genes. SNAPcSNAPc acts to assemble TFIIIB at a TATA box centered 26 base pairs upstream of the start site of transcription. It is the presence of a TATA box that specifies that the snRNA gene is transcribed by Pol III rather than Pol II. The TFIIIB for U6 snRNA transcription contains a smaller Brf1 paralogue, Brf2. TFIIIB is the transcription factor that assembles Pol III at the start site of transcription. Sequence conservation predicts that TFIIIB containing Brf2 also plays a role in promoter opening. [edit] Terminationedit Polymerase III terminates transcription at small polyTs stretch (5-6). In Eukaryotes, a hairpin loop is not required, as it is in the bacterial system.

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4 Structure of tRNAs. (a) The primary structure of yeast alanine tRNA (tRNAAla), the first such sequence determined. This molecule is synthesized from the nucleotides A, C, G, and U, but some of the nucleotides, shown in red, are modified after synthesis: D = dihydrouridine, I = inosine, T = thymine, Y = pseudouridine, and m = methyl group. Although the exact sequence varies among tRNAs, they all fold into four base-paired stems and three loops. The partially unfolded molecule is commonly depicted as a cloverleaf. Dihydrouridine is nearly always present in the D loop; likewise, thymidylate, pseudouridylate, cytidylate, and guanylate are almost always present in the TYCG loop. The triplet at the tip of the anticodon loop base-pairs with the corresponding codon in mRNA. Attachment of an amino acid to the acceptor arm yields an aminoacyl-tRNA. (b) Computergenerated three-dimensional model of the generalized backbone of all tRNAs. Note the L shape of the molecule

5 A tRNA molecule. In this series of diagrams, the same tRNA molecule—in this case a tRNA specific for the amino acid phenylalanine (Phe)—is depicted in various ways. (A) The cloverleaf structure, a convention used to show the complementary base-pairing (red lines) that creates the double-helical regions of the molecule. The anticodon is the sequence of three nucleotides that base-pairs with a codon in mRNA. The amino acid matching the codon/anticodon pair is attached at the 3′ end of the tRNA. tRNAs contain some unusual bases, which are produced by chemical modification after the tRNA has been synthesized. For example, the bases denoted Y (for pseudouridine) and D (for dihydrouridine) are derived from uracil. (B and C) Views of the actual L-shaped molecule, based on x-ray diffraction analysis. Although a particular tRNA, that for the amino acid phenylalanine, is depicted, all other tRNAs have very similar structures. (D) The linear nucleotide sequence of the molecule, color-coded to match A, B, and C.

6 Structure of a tRNA-splicing endonuclease docked to a precursor tRNA. The endonuclease (a four- subunit enzyme) removes the tRNA intron (blue). A second enzyme, a multifunctional tRNA ligase (not shown), then joins the two tRNA halves together

7 Processing of an Escherichia coli pre-tRNA. The example shown results in synthesis of tRNAtyr. The tRNA sequence in the primary transcript adopts its base-paired cloverleaf structure and two additional hairpin structures form, one on either side of the tRNA. Processing begins with the cut by ribonuclease E or F forming a new 3′ end just upstream of one of the hairpins. Ribonuclease D, which is an exonuclease, trims seven nucleotides from this new 3′ end and then pauses while ribonuclease P makes a cut at the start of the cloverleaf, forming the 5′ end of the mature mRNA. Ribonuclease D then removes two more nucleotides, creating the 3′ end of the mature molecule. With this tRNA the 3′-terminal CCA sequence is present in the RNA and is not removed by ribonuclease D. With some other tRNAs this sequence has to be completely or partly added by tRNA nucleotidyltransferase. Abbreviation: RNase, ribonuclease

8 Processing of transfer RNAs (A) Transfer RNAs are derived from pre-tRNAs, some of which contain several individual tRNA molecules. Cleavage at the 5′ end of the tRNA is catalyzed by the RNase P ribozyme; cleavage at the 3′ end is catalyzed by a conventional protein RNase. A CCA terminus is then added to the 3′ end of many tRNAs in a posttranscriptional processing step. Finally, some bases are modified at characteristic positions in the tRNA molecule. In this example, these modified nucleosides include dihydrouridine (DHU), methylguanosine (mG), inosine (I), ribothymidine (T), and pseudouridine (  ). (B) Structure of modified bases. Ribothymidine, dihydrouridine, and pseudouridine are formed by modification of uridines in tRNA. Inosine and methylguanosine are formed by the modification of guanosines.

9 Processing of tyrosine pre-tRNA involves four types of changes. A 14-nucleotide intron (blue) in the anticodon loop is removed by splicing. A 16-nucleotide sequence (green) at the 5′ end is cleaved by RNase P. U residues at the 3′ end are replaced by the CCA sequence (red) found in all mature tRNAs. Numerous bases in the stem-loops are converted to characteristic modified bases (yellow). Not all pre-tRNAs contain introns that are spliced out during processing, but they all undergo the other types of changes shown here. D = dihydrouridine; Y = pseudouridine.

10 Mechanism of splicing in pre-tRNA. First, the pre-tRNA is cleaved at two places, on each side of the intron, thereby excising the intron. The cleavage mechanism generates a 2′,3′-cyclic phosphomonoester at the 3′ end of the 5′ exon. The multistep reaction joining the two exons requires two nucleoside triphosphates: a GTP, which contributes the phosphate group (yellow) for the 3′ → 5′ linkage in the finished tRNA molecule; and an ATP, which forms an activated ligase-AMP intermediate. The 2′- phosphate on the 5′ exon is removed in the final step.

11 The structure of the aminoacyl-tRNA linkage. The carboxyl end of the amino acid forms an ester bond to ribose. Because the hydrolysis of this ester bond is associated with a large favorable change in free energy, an amino acid held in this way is said to be activated. (A) Schematic drawing of the structure. The amino acid is linked to the nucleotide at the 3′ end of the tRNA (see Figure 6-52). (B) Actual structure corresponding to boxed region in (A). These are two major classes of synthetase enzymes: one links the amino acid directly to the 3′-OH group of the ribose, and the other links it initially to the 2′-OH group. In the latter case, a subsequent transesterification reaction shifts the amino acid to the 3′ position. As in Figure 6-56, the “R-group” indicates the side chain of the amino acid.Figure 6-52Figure 6-56

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13 Helix Stacking in tRNA. The four helices of the secondary structure of tRNA () stack to form an L-shaped structure

14 Editing of Aminoacyl-tRNA. The flexible CCA arm of an aminoacyl-tRNA can move the amino acid between the activation site and the editing site. If the amino acid fits well into the editing site, the amino acid is removed by hydrolysis.

15 Amino acid activation. The two-step process in which an amino acid (with its side chain denoted by R) is activated for protein synthesis by an aminoacyl-tRNA synthetase enzyme is shown. As indicated, the energy of ATP hydrolysis is used to attach each amino acid to its tRNA molecule in a high-energy linkage. The amino acid is first activated through the linkage of its carboxyl group directly to an AMP moiety, forming an adenylated amino acid; the linkage of the AMP, normally an unfavorable reaction, is driven by the hydrolysis of the ATP molecule that donates the AMP. Without leaving the synthetase enzyme, the AMP-linked carboxyl group on the amino acid is then transferred to a hydroxyl group on the sugar at the 3′ end of the tRNA molecule. This transfer joins the amino acid by an activated ester linkage to the tRNA and forms the final aminoacyl-tRNA molecule.

16 This figure depicts the general molecular function of an aminoacyl tRNA synthetase. First, the enzyme binds the amino acid and joins it to a molecular of AMP while cleaving two phosphate groups from a molecule of ATP. Next, the enzyme binds the aminoacyl portion of this complex to an appropriate tRNA molecule while releasing the AMP molecule.

17 Hydrolytic editing. (A) tRNA synthetases remove their own coupling errors through hydrolytic editing of incorrectly attached amino acids. As described in the text, the correct amino acid is rejected by the editing site. (B) The error-correction process performed by DNA polymerase shows some similarities; however, it differs so far as the removal process depends strongly on a mispairing with the template

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19 Transcription-control elements in genes transcribed by RNA polymerase I (a) and III (b,c). Assembly of transcription-initiation complexes on these genes begins with the binding of specific general transcription factors to the control elements

20 The promoters of 5S rRNA and tRNA genes are downstream of the transcrip-tion initiation site. Transcription of the 5S rRNA gene is initiated by the binding of TFIIIA, followed by the binding of TFIIIC, TFIIIB, and the polymerase. The tRNA promoters do not contain a binding site for TFIIIA, and TFIIIA is not required for their transcription. Instead, TFIIIC initiates the transcription of tRNA genes by binding to promoter sequences, followed by the association of TFIIIB and polymerase. The TATA- binding protein (TBP) is a subunit of TFIIIB.

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