PTT 207 Biomolecular and Genetic Engineering Semester 2 2013/2014 BY: PUAN NURUL AIN HARMIZA ABDULLAH
Introduction • A central event in gene expression is the copying of the sequence of the template strand of a gene into a complementary RNA transcript. • The regulatory mechanisms that have been developed by bacteria to control transcription are complex and highly variable.
Mechanism of transcription • RNA polymerase is the enzyme that catalyzes RNA synthesis. • Using DNA as a template, RNA polymerase joins, or “ polymerizes ,” nucleoside triphosphates ( NTPs ) by phosphodiester bonds from 5' to 3'.
• In bacteria, transcription and translation are coupled ―they occur within a single cellular compartment. • As soon as transcription of the mRNA begins, ribosomes attach and initiate protein synthesis . • The whole process occurs within minutes .
Transcription and Translation are coupled in bacteria.
• Minimal requirements for gene transcription. 1. Gene promoter 2. RNA polymerase • Additional factors are required for the regulation of transcription.
1. Bacterial promoter structure • RNA polymerase binds to a region of DNA called a promoter . • Bacterial promoters are not absolutely conserved but they do have a consensus sequence .
• Conserved sequence: When nucleotide sequences of DNA are aligned with each other, each has exactly the same series of nucleotides in a given region. • Consensus sequence: there is some variation in the sequence but certain nucleotides are present at high frequency .
Bacterial promoters have 2 distinct consensus sequences.
• The σ subunit of prokaryotic RNA polymerase recognizes consensus sequences found in the promoter region upstream of the transcription start sight. • The σ subunit dissociates from the polymerase after transcription has been initiated.
Promoter strength • The relative frequency of transcription initiation . • Related to the affinity of RNA polymerase for the promoter region. • The more closely regions within the promoter resemble the consensus sequences, the greater the strength of the promoter.
2. Structure of bacterial RNA polymerase • Comprised of a core enzyme plus a transcription factor called the sigma factor ( ) . • Together they form the complete, fully functional enzyme complex called the holoenzyme .
Holoenzyme
The core enzyme • The core enzyme catalyzes polymerization. • High affinity for most DNA. • The sequence, structure, and function are evolutionarily conserved from bacteria to humans. • X-ray crystallographic studies revealed a crab claw-like shape.
Sigma factor • The sigma ( ) factor decreases the nonspecific binding affinity of the core enzyme. • Binding results in closing of the core enzyme “pincers.” • Primarily involved in recognition of gene promoters.
• In E. coli the most abundant factor is 70 . • For expression of some genes, bacterial cells use alternative factors.
Initiation of transcription Initiation consists of three stages: 1.Formation of a closed promoter complex. 2.Formation of an open promoter complex. 3.Promoter clearance.
1. Closed promoter complex • RNA polymerase holoenzyme binds to the promoter at nucleotide positions 35 and 10. • The DNA remains double-stranded. • The complex is reversible.
2. Open promoter complex • ~18 bp around the transcription start site are melted to expose the template strand DNA. • AT rich promoters require less energy to melt. • Transcription is aided by negative supercoiling of the promoter region of some genes. • The open complex is generally irreversible.
• Transcription is initiated in the presence of NTPs. • No primer is required for initiation by RNA polymerase.
3. Promoter clearance • factor does not completely dissociate; some domains are displaced. • The displaced domains allow the nascent RNA to emerge from the RNA exit channel.
Elongation • After about 9-12 nt of RNA have been synthesized, the initiation complex enters the elongation stage. • Transcription bubble - unwinds the strands at the front and rewinds them at the back
• One strand of DNA acts as the template for RNA synthesis by complementary base pairing. • Transcription always proceeds in the 5′→3′ direction.
• The catalytic site of the polymerase has both: • a substrate-binding subsite, at which the incoming NTP is bound to the polymerase and to the complementary nucleotide residue of the template, and • a product-binding subsite , at which the 3’ terminus of the growing RNA chain is positioned (figure).
• Completion of the single nucleotide addition cycle. • Shift of the active site of the RNA polymerase by one position along the template DNA.
Which moves – the RNA polymerase or the DNA? Two models • Model 1: RNA polymerase moves along and the DNA rotates. – This is the more widely accepted model. • Model 2: RNA polymerase remains stationary, and the DNA moves along and rotates.
PROOFREADING 1. Short (~5bp) backtracking motion to upstream. • The movement is directed upstream in the opposite direction to transcriptional elongation (3 ’ 5 ’) . • This backward motion carries the 3 ’ end of the nascent RNA transcript away from the enzyme active site.
PROOFREADING 2. Nucleolytic cleavage – cleave and discard the mismatched base by nuclease activity. • Occurs after a variable “pause” of the polymerase. • In its backtracked state, the polymerase is able to cleave off and discard the most recently added base(s) by nuclease activity. • In this process, a new 3 ’ end is generated at the active site, ready for subsequent polymerization onto the nascent RNA chain.
RNA Polymerase Proofreading
TERMINATION The RNA polymerase core enzyme moves down the DNA until a stop signal/terminator sequence is reached. There are 2 types of terminators: 1. Rho-dependent Requires Rho protein to stop the transcription read- through. Rho = Spider = “trap first, kill later ” Rho-independent – intrinsic terminator 2. They cause termination of transcription in the absence of any external factors.
Rho-independent
Rho-dependent
The End Thank You
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