PTT 207 Biomolecular and Genetic Engineering Semester 2 2013/2014 - - PowerPoint PPT Presentation

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

• r 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
• f 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”

2. Rho-independent – intrinsic terminator

 They cause termination of transcription in the absence of any external factors.

Rho-independent

Rho-dependent

The End

Thank You