transcription prokaryotic

8/11/2019 Transcription Prokaryotic

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Mechanisms of Transcription


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GENETIC MATERIAL

Organization Protection Replication Expression

DNA bases

RNA

Protein

Transcription

Translation


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Objectives

Understand the structure of RNA polymerases

Understand the phases of the transcription cycle

Understand the differences between transcription and replication

Understand differences between prokaryotic and eukaryotic

transcription


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Transcription is, chemically and enzymatically,

very similar to DNA replication, but there are

some important differences:

RNA is made of ribonucleotides (rather than deoxyribonuleotides)

RNA polymerase catalyzes the reaction (does not need a primer)

The synthesized RNA does not remain base-paired to the template DNAstrand

Less accurate (one in 10000,compared to one in 10000000 for replication)

Transcription selectively copies only certain parts of the genome and makesone to several hundred, or even thousand, copies of any given section of thegenome. (replication must copy the entire genome and do so once every celldivision)


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Transcription of the DNA into RNA.(in the absence of the enzymes involved)


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Multiple RNA polymerases can transcribe the same gene at the same time.A cell can synthesize a large number of RNA transcription in a short time.


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Topic 1:

RNA Polymerase and The

Transcription Cycle


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RNA Polymerases Comes in Different Forms, but

Share Many Features.

1.RNA polymerases performs essentially the same reaction in the cell,from

bacteria to humans.

2.The cellular RNA polymerases are made up of multiple subunits.

3.Bacteria have only a single RNA polymerases ,while in eukaryotic cells there

are three: RNA Pol.

4.Polis the polymerases responsible for transcribing most genesindeed,

essentially all protein-encoding genes.

5.Poland Polare each involved in transcribing specialized, RNA-encoding

genes.


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Comparison of the crystal structures of prokaryotic

and eukaryotic RNA polymerases.


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The shape of each enzyme resembles a crab claw.


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Transcription by RNA polymerases Proceeds in a Series

of steps

Initiation.

Elongation.

Termination.


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Initiation

A promoter is the DNA sequence that initially binds the RNA polymerase.

Promoter-polymerase complex undergoes structural changes required for

initiation to proceed .

The base at the transcription site unwinds and producing a bubble of

single-stranded DNA.

Transcription always occurs in a 5 to 3 direction.

Only one strand of DNA acts as a template.

For a given promoter the same strand is always transcribed


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Elongation

Once RNA polymerase synthesizes approximately 10 bases of RNA it shifts

into elongation phase

This transition requires further conformational change- tight gripping of the

template

During elongation RNA polymerase performs following functions:

A. RNA catalysis

B. Unwinds DNA in front and reanneals it behind

C. Dissociates growing RNA chain

D. Performs proofreading


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Termination

Once the polymerase has transcribed the length of the gene (or genes), it must

stop and released the RNA product. This step is called termination.

In some cells, termination occurs at the specific and well-defined DNA

sequences called terminators. Some cells lack such termination sequences.


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Transcription Initiation Involves three Defined

Steps

The initial binding of polymerase to promoter to form what is called a closed

complex: DNA double stranded and enzyme bound to it

The closed complex undergoes a transition to the open complex: DNA strand

separates over a distance of around 14 bp around the start site to form a

transcription bubble First two nucleotides brought into the active site, aligned to the template and

then joined together. This followed by enzyme movement on the template

strand

Incorporation of around first 10 nucleotides is inefficient and often at this stage

enzyme releases short transcripts. Once a enzyme gets further than the 10 bp it starts synthesizing RNA

efficiently. This process is also known as promoter escape


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Topic 2:

The Transcription Cycle in

Bacteria


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Bacteria Promoters Vary in Strength and Sequence,

but Have Certain Defining Features

The bacteria core RNA polymerases can, in principle, initiate transcription

at any point on a DNA molecule.

In cells, polymerases initiate transcription only at promoters.

An initiation factor called that converts core enzyme into the form that

initiates only at promoter. That form of the enzymes is called the RNA

polymerase holoenzyme.

In the case ofE.coli, the predominant is called 70. Promoter recognized by

70 contains two conserved sequences (-35 and 10 regions/elements)

separated by a non-specific stretch of 17-19 nt. Position +1 is the transcription start site


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Features of bacteria promoters. Various combination of bacteria promoter elements are shown.

a. 70 promoters contain recognizable 35 and10 regions, but the sequences are not identical.

b. UP-element is an additional DNA elements that increases polymerase binding by providing the

additional interaction site for RNA polymerase.

c. Another class of s70 promoter lacks a35 region and has an extended 10 element

compensating for the absence of35 region.


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The Factor Mediate Binding of Polymerase to the

Promoter

Regions of Those regions factor that

recognize specific regions of the promoter

are indicated by arrows. Region 2.3 isresponsible for melting the DNA.

2 binds to -10 and 4 binds to -35

4- helix turn helix, 2- helix

-35 in mere binding

-10 binding and transcription initiation,

DNA melting initiated in this region

3 binds to extended -10


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and subunits recruit RNA polymerases core Enzyme to the promoter. The C-domain of the

subunit ( CTD) Recognize the UP-element, while region 2 and 4 recognize the -10 and -35regions respectively.

UP-element not recognized by , instead by carboxyl terminal domain of

subunit of RNA polymerase


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Transition to the Open Complex Involves Structural

Changes in RNA Polymerase and in the Promoter DNA

Transition from closed to open complex (also known as isomerization)

requires DNA melting between positions -11 and +3. Isomerization is

independent of ATP hydrolysis

After Isomerization transcription is essentially irreversible; Formation of closed

complex is reversible


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Two striking structural changes during Isomerization:

A. Pincers at the front end of the enzyme clamp down tightly on the downstream DNA

B. factor N-terminal region (region 1.1) lies in the path of DNA in closed complex.

During isomerization this region shifts some 50 A and gets away from the path of

incoming DNA. This ensures that DNA is accessible to the active centre.


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Transcription is Initiated by RNA Polymerase without

the Need for a Primer

DNA polymerase can only extend an existing polynucleotide chain.

However, RNA polymerase can initiate a new RNA chain on a DNA templateand does not need a primer.


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RNA Polymerase Synthesizes Several Short RNAs

Before Entering the Elongation Phase

Abortive initiation: enzyme synthesizes short RNA molecules of less than 10

nucleotides in length.

factor region 3.2 lies in the middle of RNA exit channel in the open complex

and to make RNAs longer than 10 nucleotides, this region should be ejected

from that location. This process of ejecting sometimes require many attempts.

After ejection of region 3.2, factor becomes weakly associated with the

elongating enzyme and it is often lost from the elongating complex.


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The Elongating Polymerase is a Processive Machine that

Synthesizes and Proofreads RNA

DNA separated at catalytic cleft, NT and T strands follow different paths, before

exiting via their respective channels to reanneal. In this process RNA gets

synthesized using nucleotides and template strand as guide.

Only 8-9 nucleotides of growing RNA chain remain base paired; remainder of

the RNA chain is peeled off and exits through RNA exit channel.

Two proofreading functions:

Pyrophosphorolytic editing: simple back reaction to catalyze removal of an incorrectly

inserted ribonucleotide, by incorporation of PPi. This is then replaced by a correctnucleotide.

Hydrolytic editing: Polymerase backtracks by one or more nucleotides and cleaves

the RNA product, thereby removing the error-containing sequence.


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Transcription is terminated by signals within the RNA

sequence

Terminators: the sequence that trigger the elongating polymerases to

dissociate the DNA and release the RNA chain it has made.

In bacteria, terminators come in two type: Rho-independent (also called as

intrinsic terminator) and Rho-dependent.

Rho-independent causes polymerase to terminate without involvement of other

factors, whereas Rho-dependent termination need Rho protein.


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Transcription termination. Shown is a model for howthe Rho-independent terminator might work.

transcription prokaryotic

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