Transcription

Transcription, Promoter, Promoter Structure, RNA polymerase, Sigma factor, Steps of transcription, Trasncriptional Initiation, Transcription Elongation, Transcriptional Termination, Mechanisms of termination, Rho dependent termination , Rho indepenent termination, Transcription in prokaryotes, Transcription in Eukaryotes.

1. TRANSCRIPTION BY HASNAT TARIQ

2. DNA TRANSCRIPTION It is a process in which RNA is synthesized by using DNA as a template. DNA (Double Stranded) RNA ( Single Stranded) RNA Polymerase is the key enzyme in transcription process. 2

3. RNA Polymerase Structure in Prokaryotes In 1969, the polypeptides that make up the E. coli RNA polymerase had been identified by SDS polyacrylamide gel electrophoresis (SDS-PAGE). Two very large subunits: beta (β) and beta-prime (β’) , with molecular masses of 150 and 160 kD . The other RNA polymerase subunits visible on this gel are called sigma (s) and alpha (a), with molecular masses of 70 and 40 kD . Another subunit, omega (ω), with a molecular mass of 10 kDa is not detectable here, but was clearly visible in urea gel electrophoresis experiments. In 1969, the polypeptides that make up the E. coli RNA polymerase had been identified by SDS polyacrylamide gel electrophoresis (SDS-PAGE). Two very large subunits: beta (β) and beta-prime (β’) , with molecular masses of 150 and 160 kD . The other RNA polymerase subunits visible on this gel are called sigma (s) and alpha (a), with molecular masses of 70 and 40 kD . Another subunit, omega (ω), with a molecular mass of 10 kDa is not detectable here, but was clearly visible in urea gel electrophoresis experiments. 3

4. Continue.. β': The β' subunit is the largest subunit , and is encoded by the rpoC gene. β: The β subunit is the second-largest subunit , and is encoded by the  rpoB  gene α I  and α II : The α subunit is the third-largest subunit  . ω: The ω subunit is the smallest subunit. 4

5. Sigma ( σ ) as a Specificity Factor Sigma factors are subunits of all bacterial RNA polymerases. They are responsible for determining the specificity of promoter DNA binding and control how efficiently RNA synthesis (transcription) is initiated. The first sigma factor discovered was the sigma70 ( σ 70 ) of the highly studied bacterium  Escherichia coli .   A core polymerase (with subunit structure α2ββ′) can transcribe DNA into RNA inefficiently and nonspecifically. With sigma factor, it can bind to core forming a holoenzyme (α2ββ′σ) that is capable of specific engagement with duplex DNA at the beginning of genes (promoters) as well as efficient initiation of transcription.     5

6. Promoters The polymerase binding sites are called promoters. Present at the upstream of the coding region of DNA. Transcription that begins at promoters in vitro is specific and mimics the initiation that would occur in vivo. Thus, operates by directing the polymerase to initiate at specific promoter sequences. Sigma stimulates tight binding between RNA polymerase and promoter. 6

7. Binding of RNA Polymerase to Promoters The holoenzyme finds two kinds of binding sites, tight binding sites and loose ones. On the other hand, the core polymerase is capable of binding only loosely to the DNA. a) Promoter search The holoenzyme binds and rebinds loosely to the DNA, searching for a promoter. b) Closed promoter complex formation The holoenzyme as found a promoter and has bound loosely, forming a closed promoter complex. c) Open promoter complex formation The holoenzyme has bound tightly, melting a local region of DNA and forming an open promoter complex. 7

8. Prokaryotic Promoters Promoters in prokaryotic organisms are two short DNA sequences located at the -10 (10bp 5' or upstream) and -35 positions from the transcription start site. The Pribnow box (TATAAT) is located at the -10 position and is essential for transcription initiation. The -35 position , simply titled the -35 element , typically consists of the sequence TTGACA and this element controls the rate of transcription.  8

9. Eukaryotic Promoters Eukaryotic Promoters are complex and diverse . 9

10. Enhancers Enhancer DNA sequences bind transcription factors with special protein called enhancer-binding proteins which increase the rate of transcription. Enhancer sequences may be at a distance of kilobases away from the gene they influence. An enhancer complex may interact with promoter complexes by bringing the sites into direct contact, may be by formng a loop-like structure. 10

11. Steps in Transcription Initiation Elongation Termination Initiation Elongation Termination 11

12. 1. Initiation Stages of transcription initiation (a) RNA polymerase binds to DNA in a closed promoter (b) The s-factor stimulates the polymerase to convert the closed promoter complex to an open promoter complex. (c) The polymerase incorporates the first 9 or 10 nt into the nascent RNA. Some abortive transcripts are pictured at left. (d) The polymerase clears the promoter and begins the elongation phase. The s-factor may be lost at this point or later, during elongation. 12

13. 2. Elongation After initiation of transcription is accomplished, the core continues to elongate the RNA, adding one nucleotide after another to the growing RNA chain. 13

14. Core Polymerase Functions in Elongation The core polymerase contains the RNA synthesizing machinery, so the core is the central player in elongation. Beta (β) and beta-prime (β’) subunits are involved in phosphodiester bond formation, that these subunits also participate in DNA binding. (α ) subunit has several activities, including assembly of the core polymerase. 14

15. 3. Termination RNA synthesis will continue along the DNA template strand until the polymerase encounters a signal that tells it to stop, or terminate, transcription. 15

16. Termination in Eukaryotes 16

17. Termination in Prokaryotes In prokaryotes, there are two different mechanisms of termination. rho-independent rho-dependent. 17

18. Rho-independent Termination The rho-independent terminator is the more simple of the two systems and as a result is also called simple termination. The rho-independent signal is found on the DNA template strand and consists of a region that contains a section that is then repeated a few base pairs away in the inverted sequence. 18

19. Rho-dependent Termination The rho-dependent terminator received its name because it is dependent on a specific protein called a rho factor . The rho factor is thought to bind to the end of the RNA chain and slide along the strand towards the open complex bubble. When the factor catches the polymerase, it causes the termination of transcription. The mechanism of this termination is unclear, but the rho factor could in some way pull the polymerase complex off of the DNA strand. 19

20. THANK YOU 20