RNA polimerase RNA polimerase

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transcription: synthesize of RNA by RNA polimerase.transcription is started in promotor and ended up in terminatorThe sequence which exists before the startpoint is called as the upstream (-), while thesequence 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 RNApolymerase

In the bubble area : RNA polymerase is started to unveil the DNA,synthesize RNA and re-twist the DNA

Daerah gelembung DNA

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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.

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Transcription is divided in to 4 steps : Template recognition, initiation, elongation andtermination

RNA polimerase

RNA polymerase in Bacteria

In E. coli there is only one type of mRNA, tRNA, rRNA in 1

cell, there will be 7000 molecules of RNA pol,

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

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Rifampisin inhibits initiation, streptolydigine

inhibits the elongation. Moreover, the two

antibitoics disrupt the subunitfor 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.

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Gene MW(KDa)KDa)

Purpose Sequence

– 10

Sequence

– 35

rpoD 700 general TATAA TTGAC

rpoH / 32 / H 32 Heatshock CCCC CCCTT

HrpoN / 54 / N 54 Nitrogen TTGC CTGGN

NFliA / 28 / F 28 flagella GCCG CTAAA

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RNA pol. do not possess nuclease activity,

therefore the correction of the forming

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

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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.

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rho independent terminationHairpin-shaped terminator, in secondary structure and also occupied with ~ 6U. 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.

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The recognized RNA sequence is C-rich and lackof G residues. The efficiency of terminator isvalued up along with the extension of C-rich/lackG 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.

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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 asborder

• By doing the post-transcription, the rRNAmolecule will be dissociated to each other andalso to tRNA.

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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.

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The Post Transcription of rRNA

tRNa Process

tRNA is derived from 80 nucleotides, in 4-stalk

of its secondary structure. The former bases are

called as kleebalt structure. tRNA mostly

consists of modified bases and CCA sequence

on 3-end which is utilized as the connection to

amino acid.

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Pre-tRNA

tRNA is formed by only one operon (groups of genes which is squeezedby only one promotor-terminator sequence), therefore the transcription willgenerate a long RNA which consist of lots of tRNA.

Post Transcription oftRNA

The cutting of pre-tRNAto individual tRNA

The adding of ACC incertain 3’-endsequence

The slicing of intron incertain tRNA

The modification of thecertain base

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Ujung 3’ACC,penempelanasam amino

SimpulTC

SimpulVariasi

SimpulKodon

SecondaryStructure of tRNA

Simpul D

Modifiedbase

Original BaseThe modifiedbase of tRNA

A

Modified base

i6A m’A

D

m’G

I

s2C

Gm

Im

T

m5C

G

U

C