1. RNA polimerase 9/21/2010 transcription: synthesize of RNA by RNA polimerase. transcription is started in promotor and ended up in terminator The sequence which exists before the startpoint is called as the upstream (-), while the sequence after the startpoint is stated as the downstream (+). transcription yields single strand RNA The length of transcription bubble is ~ 18 pb, but the hybrid area is shorter (approximately ~ 12 pb). The bubble is maintained during the transcription process by RNA polymerase In the bubble area : RNA polymerase is started to unveil the DNA, synthesize RNA and re-twist the DNA Daerah gelembung DNA 1

2. 9/21/2010 2 Transcription Step: 1. Recognizing the template  binding of the RNA pol to the DNA double helix  the DNA strand is separated. The sequence that is recognized by RNA pol is called as promotor. transcription is the first step of the gene expression 2. Initiation is the synthesize of the first nucleotide bond in RNA strand. Initiation step is the regulation step which determine the transcription of one gene. The enzyme lays still in promotor site during the synthesize of nine foremost nucleotides. 3. Elongation is the movement of RNA pol enzyme along the DNA strand. The elongation involves the movement of the transcription bubble due to the alteration of DNA structure, where the template strand is joined to the nascent RNA in the growing site. 4. Termination involves the recognition of a certain site where the adding of new nucleotide is no longer happened. Sequence that is needed is called as terminator.

3. 9/21/2010 Transcription is divided in to 4 steps : Template recognition, initiation, elongation and termination RNA polimerase RNA polymerase in Bacteria  In E. coli there is only one type of mRNA, tRNA, rRNA  in 1 cell,therewillbe7000moleculesofRNApol, approximately. Holoenzyme, 480 kDa consists of:  2  ’  Core – enzyme :  2  ’ Sigma factor is only involved in early transcription. After 8-9 bases are constructed, the sigma factor is released and the core-enzyme will take up the elongation process  faktor  is only for initiation 3

4. 9/21/2010 Rifampisininhibitsinitiation,streptolydigine inhibitstheelongation.Moreover,thetwo antibitoics disrupt the  subunit  for the binding of nucleotide Heparin binds the  ’ and inhibits the transcription in vitro. Heparin competes the DNA to bind the polymerase  ’ for the binding to DNA matrix  for the development of core – enzyme. 4

5. Gene MW (KDa)KD a) Purpose Sequence – 10 Sequence – 35 rpoD700generalTATAATTGAC rpoH /  32 /  H32HeatshockCCCCCCCTT HrpoN /  54 /  N54NitrogenTTGCCTGGN NFliA /  28 /  F28flagellaGCCGCTAAA 9/21/2010 RNA pol. do not possess nuclease activity, thereforethecorrectionoftheforming polynucleotide can not be maintained. Core enzyme can not distinguish the promotor sequence to the other sequence in the DNA sequence. Sigma factor changes the ability of the enzyme so that the enzyme may recognize the specific binding site Sigma Factor in E. coli 5

6. 9/21/2010 6 Promotor recognition The DNA suquence that is functional for the recognition such as promotor and regulation site is differ in structure with the other sequence that is being translated or transcribed. There are two type of promotor: strong promotors (promotor sequence which is frequently transcribed, every 2 seconds in E. coli) and weak promotor (every 10 minutes) In bacteria, the minimal size of promotor sequence is 12 pb, which un- necessarily close to each other. The terminator in E.coli is differentiated regarding to the requirement of the other factor: 1. Rho independent = intrinsic terminator happens without the other factor in vitro. 2. Rho dependent requires the rho factor in vitro. Based on the mutation analysis, the rho factor is having a role to terminate the transcription in vivo.

7. 9/21/2010 7 rho independent termination Hairpin-shaped terminator, in secondary structure and also occupied with ~ 6 U. The length is vary, generally contain of GC rich sequence. Rho dependent termination The addition of Rho factor may affect the RNA pol to terminate the transcription in a certain site by generating a RNA molecule with a unique 3- end sequence Rho is an essential protein in E. coli, although its genomic structure rarely has the rho dependent terminator. Commonly this type of terminator is occurred in phage.

8. 9/21/2010 8 The recognized RNA sequence is C-rich and lack of G residues. The efficiency of terminator is valued up along with the extension of C-rich/lack G area (rho-dependent terminator) How the rho factor works? Rho factor is only involved in termination. The factor is a protein (46 kDa) and probably active as hexamer (275 kDa). Rho has RNA-dependent ATPase activity, needs the presence of polyribonucleotide > 50 bases. It has been suggested that the rho binds the RNA.

9. 9/21/2010 9 The Modification of rRNA Post--Transcription In E. coli, rRNA is formed by seven operons {rrnA, rrnB, rrnC,..rrnH} In E. coli, pre-rRNA contains of rRNA5S, rRNA23S, rRNA16S, and numbers of tRNA as border By doing the post-transcription, the rRNA molecule will be dissociated to each other and also to tRNA.

10. 9/21/2010 10 rRNA Process The seven operons of rRNA in E. coli has copy of the 3 rRNA genes. The primer transcript is 5500 nucleotide longer, 16S-rRNA sequence is existed in 5’-end, followed by 1 or 2 tRNA, 23S-rRNA, 5S-rRNA, moreover in certain operons, 1 or 2 tRNA in 3’-end sequence.

11. 9/21/2010 The Post Transcription of rRNA tRNa Process tRNA is derived from 80 nucleotides, in 4-stalk of its secondary structure. The former bases are calledaskleebaltstructure.tRNA mostly consists of modified bases and CCA sequence on 3-end which is utilized as the connection to amino acid. 11

12. 9/21/2010 12 Pre-tRNA tRNA is formed by only one operon (groups of genes which is squeezed by only one promotor-terminator sequence), therefore the transcription will generate a long RNA which consist of lots of tRNA. Post Transcription of tRNA The cutting of pre-tRNA to individual tRNA The adding of ACC in certain 3’-end sequence The slicing of intron in certain tRNA The modification of the certain base

13. 9/21/2010 13 Ujung 3’ACC, penempelan asam amino Simpul T  C Simpul Variasi Simpul Kodon Secondary Structure of tRNA Simpul D Modified base Original Base The modified base of tRNA A Modified base i6A m’A D  m’G I s 2 C Gm I m T m 5 C GUCGUC