RNA POLYMERASE

INTRODUCTION

Acts as an intermediary, carrying genetic information from the DNA to the

machinery of protein synthesis.

Synthesizes all types of RNA in the cell. Inhibited by rifampicin and

Actinomycin D binds to the DNA preventing transcription.

Performs the same reaction in all cells, from bacteria to humans. Made up of

multiple subunits.

In cells , RNAP is necessary for constructing RNA chains using DNA genes

as templates, a process called transcription.

RNAP is a nucleotidyl transferase that polymerises ribonucleotides at the 3’

end of an RNA transcript.

PURIFICATION

RNA Polymerase can be isolated

By a phosphocellulose column

By glycerol gradient centrifugation

By a DNA column

By an ion chromatography column

HISTORY

RNAP was discovered independently by Charles Loe, Audrey Stevens, and Jerard

Hurwitz in 1960. By this time, one half of the 1959 Noble Prize in Medicine had been

awarded to Severo ochoa for the discovery of what was believed to be RNAP.

The 2006 Nobel Prize in Chemistry was awarded to Roger D. Kornberg for creating

detailed molecular images of RNA polymerase during various stages of the transcription

process.

Using Yeast, Kornberg identified the role of RNA polymerase II and other proteins in

transcribing DNA, and he created three-dimensional images of the protein cluster using

X-Ray crystallography. Polymerase II is used by all organisms with nuclei, including

humans, to transcribe DNA.

HISTORY

Kornberg's research group at Stanford later succeeded in the development of a faithful

transcription system from baker’s yeast, a simple unicellular eukaryote, which they

then used to isolate in a purified form all of the several dozen proteins required for the

transcription process.

Using this system, Kornberg made the major discovery that transmission of gene

regulatory signals to the RNA polymerase machinery is accomplished by an additional

protein complex that they dubbed Mediator. The discovery of Mediator is therefore a

true milestone in the understanding of the transcription process.

He devoted two decades to the development of methods to visualize the atomic structure

of RNA polymerase and its associated protein components.

HISTORY

Kornberg took advantage of expertise with lipid membranes gained from his graduate

studies to devise a technique for the formation of two-dimensional protein crystals on

lipid bilayers. These 2D crystals could then be analyzed using electron microscopy to

derive low-resolution images of the protein's structure. Eventually, Kornberg was able to

use X-Ray crystallography to solve the 3- Dimensional structure of RNA polymerase

at atomic resolution.

He has recently extended these studies to obtain structural images of RNA polymerase

associated with accessory proteins.

PRODUCTS OF RNAP

Messenger RNA

Non-coding RNA  or "RNA genes

Transfer RNA

Ribosomal RNA

Micro RNA

Catalytic RNA (Ribozyme)

RNAP accomplishes de novo synthesis. It is able to do this because

specific interactions with the initiating nucleotide hold RNAP rigidly

in place, facilitating chemical attack on the incoming nucleotide-

RNAP prefers to start transcripts with ATP (followed by GTP, UTP,

and then CTP). In contrast to DNAP, RNAP includes

helicase activity, no separate enzyme is needed to unwind DNA.

Prokaryotic Eukaryotic

Bacterial Archaeal RNAP Ⅰ RNAP Ⅱ RNAP Ⅲ

Core Core (Pol )Ⅰ (Pol )Ⅱ (Pol )Ⅲ

β‘ A’/A” RPC1 RPB1 RPC1

β B RPA2 RPB2 RPC5

α’ D RPC5 RPB3 RPC5

α“ L RPC9 RPB11 RPC6

ω K RPB6 RPB6 RPB6

[+6 other] [+9 others] [+7 others] [+11 others]

Transcription by RNA Polymerase

THREE TYPES OF RNA POLYMERASE

RNA polymerase I is located in the nucleolus and synthesizes 28S, 18S, and 5.8S

rRNAs.

RNA polymerase II is located in the nucleoplasm and synthesizes hnRNA/mRNA

and some snRNA.

RNA polymerase III is located in the nucleoplasm and synthesizes tRNA, some

snRNA, and 5S rRNA.

Transcription factors (such as TFIID for RNA polymerase II) help to initiate

transcription.

Prokaryotic Transcription Unit

EXPRESSION OF A PROKARYOTIC GENE

The mRNA produced by the gene- monocistronic message. It is transcribed

from a single gene and codes for only a single protein.

Cistron- another name for a gene.

Some bacterial operons produce polycistronic messages. In these cases,

related genes grouped together in the DNA are transcribed as one unit.

The mRNA in this case contains information from several genes and

codes for several different proteins

Prokaryotic Polycistronic Message Codes for Several Different Proteins

Upstream elements: quite varied in number and can be orientation-

independent (but relatively position-dependent) & recognized by other TFs

(relatively gene-specific) that participate in initiation at smaller sub-sets of

promoters.

1. GC box (GC rich)

2. CAAT box (5’-CCAAT-3)

e.g., GC boxes bind the TF Sp1, while CCAAT boxes bind CTF

Eukaryotic Transcription Unit

ENHANCERS AND SILENCERS

Enhancers: Increase the amount of Transcription from a nearby

promoter (core + upstream elements)

Silencers: Decrease amount of Transcription from nearby promoters

Initially Defined as being “Position and orientation independent”

Found upstream, within, or downstream of genes

Function in either orientation.

RESULT OF THE TRANSCRIPTION CYCLE

RNA polymerase dissociates the RNA transcript from the DNA as it

is transcribed.

Multiple RNA polymerase can transcribe the same gene at the same

time

A cell can synthesize a large number of RNA transcripts in a short

time.

RESEARCH ON RNAP III TRANSCRIBES HUMAN

MICRORNAS

Glen M Borchert et al; demonstrated that mammalian microRNA expression requires RNAP II.

However, the transcriptional requirements of many miRNAs remain untested.

Genomic analysis of miRNAs in the human chromosome19 miRNA cluster (C19MC) revealed that

they are interspersed among Alu repeats.

Because Alu transcription occurs through RNAP III recruitment, and they found that Alu elements

upstream of C19MC miRNAs retain sequences important for Pol III activity, they tested the promoter

requirements of C19MC miRNAs.

Chromatin immunoprecipitation and cell-free transcription assays showed that Pol III, but not Pol II, is

associated with miRNA genomic sequence and sufficient for transcription

The mature miRNA sequences of approximately 50 additional human miRNAs lie within Alu and other

known repetitive elements.

RNAP I–specific subunits promotepolymerase clustering to enhance the rRNA gene

transcription cycle Benjamin Albert et al; showed that the Rpa49 and Rpa34 Pol I subunits, which

do not have counterparts in Pol II and Pol III complexes, are functionally

conserved using heterospecific complementation of the human and

Schizosaccharomyces pombe orthologues in Saccharomyces cerevisiae.

Deletion of RPA49 leads to the disappearance of nucleolar structure, but nucleolar

assembly can be restored by decreasing ribosomal gene copy number from 190 to

25.

Statistical analysis of Miller spreads in the absence of Rpa49 demonstrates a

fourfold decrease in Pol I loading rate per gene an decreased contact between

adjacent Pol I complexes.

RNAP II–TFIIB STRUCTURE AND

MECHANISM OF TRANSCRIPTION INITIATION

Michael Thomm et al; presented the crystal structure of the complete Pol II–B complex at

4.3A° resolution, and complementary functional data. The mechanism of transcription

initiation, including the transition to RNA elongation. Promoter DNA is positioned over the

Pol II active centre cleft with the ‘B-core’ domain that binds the wall at the end of the cleft.

DNA is then opened with the help of the ‘B-linker’ that binds the Pol II rudder and clamp

coiled-coil at the edge of the cleft. The DNA template strand slips into the cleft and is

scanned for the transcription start site with the help of the ‘B-reader’ that approaches the

active site.

Synthesis of the RNA chain and rewinding of upstream DNA displace the B-reader and B-

linker, respectively, to trigger B release and elongation complex formation.

FIVE CHECKPOINTS MAINTAINING THE FIDELITY OF

TRANSCRIPTION BY RNAP IN STRUCTURAL AND

ENERGETIC DETAILS

The effects of different active site NTPs in both open and closed trigger loop (TL)

structures of RNAPs are compared. Unfavorable initial binding of mismatched

substrates in the active site with an open TL.

The closing motion of the TL, required for catalysis, is hindered by the presence of

mismatched NTPs. Mismatched NTPs- conformational changes in the active site, which

perturb the coordination of Mg ions and affect the ability to proceed with catalysis.

Structural perturbations in the template DNA and the nascent RNA in the presence of

mismatches- hinder nucleotide addition and provide the structural foundation for

backtracking followed by removing erroneously incorporated nucleotides during

proofreading.