Peter John M.Phil, PhD Atta-ur-Rahman School of Applied Biosciences (ASAB) National University of Sciences & Technology (NUST)

DNA Transcription Transcription produces an RNA chain identical in sequence with one strand of the DNA, sometimes called the coding strand. This strand is made 5 ‘----3 ' and is complementary to (i.e., it base-pairs with) the template, which i s 3 ‘---5'. The RNA like strand therefore is called the non template strand

Transcription Unit A transcription unit is a sequence of DNA transcribed into a single RNA, starting at the promoter and ending at the terminator.

DNA Transcription Terminology Promoter Seq: The sequence of DNA needed for RNA polymerase to bind to the template and accomplish the initiation reaction. Start Point: Ist base where transcription start & then termination occur Transcription Unit: Region b/w promoter to termination Up Stream seq: Seq prior to the start point (-ve) Down Stream Seq: Seq after the transcribe seq (+ve) Primary Transcript: Immediate product of transcription

DNA Transcription Terminology RNA synthesis in 5’-------3’ direction Replication Rate: 800bp/sec Transcription Rate: 40bp/sec Translation Rate: 15 AA/sec RNA Pol. Create a transcription bubble where it binds to a promoter.

Start Point & Termination

Transcription Occurs by Base Pairing in a “Bubble" of Unpaired DNA RNA polymerase separates the two strands of DNA in a transient "bubble" and uses one strand as a template to direct synthesis of a complementary sequence of RNA. The bubble is 12 to 14 bp, and the RNA-DNA hybrid within the bubble is 8 to 9 bp.

Transcription Bubble Transcription takes place in a bubble, in which RNA is synthesized by base pairing with one strand of DNA in the transiently unwound region. As the bubble progresses, the DNA duplex re-forms behind it, displacing the RNA in the form of a single polynucleotide chain.

Transcription Bubble RNA Pol. Coding (sense) strand 5’ 3’ Template (antisense) strand Coding (sense) strand RNA Pol.

Transcription Bubble RNA Pol. Coding (sense) strand 5’ 3’ RNA Pol. 3’ 5’ Template (antisense) strand

Transcription Bubble

Template recognition Initiation Elongation Termination Transcription Stages Template recognition Initiation Elongation Termination

Transcription Stages Template recognition: begins with the binding of RNA polymerase to the double-stranded DNA at a promoter to form a "closed complex". Then the strands of DNA are separated to form the "open complex“ Initiation: describes the synthesis of the first nucleotide bonds in RNA. The enzyme remains at the promoter while it synthesizes the first ~9 nucleotide bonds.

Transcription Stages Elongation: Elongation involves the movement of the transcription bubble by a disruption of DNA structure, in which the template strand of the transiently unwound region is paired with the nascent RNA at the growing point. Termination: Involves recognition of the point at which no further bases should be added to the chain. To terminate transcription.

Transcription Stages The enzyme binds to the promoter and melts DNA and remains stationary during initiation; moves along the template during elongation; and dissociates at termination.

Transcription Stages

Bacterial RNA Polymerase Consists of Multiple Subunits Bacterial RNA core polymerases are 400 kD multi subunit complexes with the general structure Catalysis derives from 13' subunits. About 13,000 RNA polymerase molecules are present in an E. coli cell, although the precise number varies with the growth conditions

Bact RNA Polymerase 2,000-5,000 are synthesizing RNA at any one time The complete enzyme or holoenzyme in E.Coli has a M.Wt. ~465 kDa

Bacterial RNA Polymerase Sub units

Bacterial RNA Polymerase Sub units

Bacterial RNA Polymerase Sub units

RNA Polymerase Core & Sigma Factors The Holoenzyme can be divided into 2 components Core Enzyme: The core enzyme has four polypeptide subunits: alpha (a), beta (b), beta' (b'), and omega (w) in the stoichiometry a2bb'w Core enzyme has the ability to synthesis RNA but can’t initiate transcription It can’t distinguished b/w promoter & other DNA seq

RNA Polymerase Core & Sigma Factors Sigma Factor: It directs RNA polymerase to the promoter and ensures that transcription is initiated only where it is supposed to be initiated. Sigma factor release when chain reaches 8-9 bases

Core & Sigma Factors

RNA Polymerase Core & Sigma Factors

RNA Pol Conformational Changes RNA Pol initially binds to DNA it covers 70-80 bases (starting -55 to +20) Then shape of RNA Pol changes from initiation to elongation, which cover ~ 60 bases

RNA Pol Conformational Changes

RNA Pol Function

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