Rna polymerase

RNA Polymerase
Introduction
Purification
History
PRODUCTS OF RNAP
Messenger RNA
Non-coding RNA  or "RNA genes
Transfer RNA
Ribosomal RNA
Micro RNA
Catalytic RNA (Ribozyme)
prokaryotic and eukaryotic
Transcription by RNA Polymerase
TYPES OF RNA POLYMERASE
Type I
Type II
Type III
Prokaryotic Transcription Unit
EXPRESSION OF A PROKARYOTIC GENE
Prokaryotic Polycistronic Message Codes for Several Different Proteins
Eukaryotic Transcription Unit
ENHANCERS AND SILENCERS
RESULT OF THE TRANSCRIPTION CYCLE
RNAP III TRANSCRIBES HUMAN MICRORNAS
RNAP I–specific subunits promote�polymerase clustering to enhance the rRNA gene�transcription cycle
RNAP II–TFIIB STRUCTURE AND�MECHANISM OF TRANSCRIPTION INITIATION
FIVE CHECKPOINTS MAINTAINING THE FIDELITY OF�TRANSCRIPTION BY RNAP IN STRUCTURAL AND�ENERGETIC DETAILS

1. RNA POLYMERASE

2. INTRODUCTION Acts as an intermediary, carrying genetic information from the DNA to the machinery of protein synthesis. Sy nthesizes 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.

3. PURIFICATION RNA Polymerase can be isolated By a phosphocellulose column By glycerol gradient centrifugation By a DNA column By an ion chromatography column

4. 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.

5. 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.

6. 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.

7. PRODUCTS OF RNAP Messenger RNA Non-coding RNA  or "RNA genes Transfer RNA Ribosomal RNA Micro RNA Catalytic RNA (Ribozyme)

8. 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.

9. 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]

10. Transcription by RNA Polymerase

11. 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.

12. Prokaryotic Transcription Unit

13. 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

14. Prokaryotic Polycistronic Message Codes for Several Different Proteins

15. 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

16. Eukaryotic Transcription Unit

17. 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.

18. 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.

19. 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.

20. RNAP I–specific subunits promote polymerase 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.

21. 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.

22. 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.