transcription of prokaryotes(bhanu shrestha)

1. BHANU SHRESTHA MSC MICROBIOLOGY(FIRST SEMESTER) TRANSCRIPTION IN PROKARYOTES

2. Background Components Aspects Steps: Initiation Elongation Termination CONTENTS

3. Central Dogma- Within the cell, genetic information is transferred from DNA to mRNA (transcription) and then to protein (translation); proposed by Francis Crick. Synthesis of RNA under the direction of DNA is called Transcription. RNA product has a sequences complementary to the DNA template directing its synthesis. BACKGROUND

5. Transcription generates 3 kinds of RNA: Messenger RNA(mRNA) Transfer RNA(tRNA) Ribosomal RNA(rRNA) Protein synthesis takes place in Ribosomes. Before protein synthesis can proceed the coding of DNA must first be transferred to a substance that passes information from the DNA in the nuclear region to the ribosome in the cytoplasm. The substance is mRNA. But since there is no nucleus in Prokaryotes transcription and translation can be directly coupled. Ribosomes attach to mRNA as it is being synthesized from the DNA template.

7. DNA TRANSCRIPTION mRNA Ribosome Prokaryotic cell Polypeptide LE 17-3-2 Prokaryotic cell

8. Basic components for transcription dsDNA with a promoter RNA polymerase rNTPs(ribonucleotidestriphosphates) ATP, CTP, GTP, UTP Transcription is the process or step in which a single stranded mRNA is catalyzed by the enzyme RNA polymerase. Activated nucleotides are required as substrate for this enzymes.

9. Elongation Non-template strand of DNA RNA nucleotides RNA polymerase 3¢ 3¢ end 5¢ LE 17-7 Direction of transcription (“downstream”) 5¢ Template strand of DNA Newly made RNA

10. Promoter: DNA sequence where RNA polymerase binds to transcribe the gene Transcription start site: the nucleotide where RNA pol initiates transcription Transcription unit: the transcribed DNA • Structure of a gene

11. Transcription unit Promoter 5¢ 3¢ 3¢ 5¢ DNA Start point RNA polymerase LE 17-7

12. Transcription involves following 3 aspects:- • Enzymatic Synthesis of RNA • The signals that determine at what points on a DNA molecule transcription starts and stops • The types of transcription products and how they are converted to the RNA molecules needed by the cell Aspects of Transcription

13. The three stages of transcription: • Initiation • Elongation • Termination Synthesis of an RNA Transcript

14. Promoter Transcription unit 5 3 3¢ 5¢ DNA Start point RNA polymerase Initiation 5¢ 3¢ 5¢ 3¢ Template strand of DNA RNA tran- script Unwound DNA Elongation Rewound DNA 5¢ 3¢ 3¢ 3¢ 5¢ 5¢ RNA transcript Termination LE 17-7 5¢ 3¢ 3¢ 5¢ 5¢ 3¢ Completed RNA transcript

15. RNA synthesis: 5' to 3' direction(new nucleotide added to 3' end of growing chain) at rate of 40 nucleotides per second at 37⁰C • RNA polymerase unwinds double helix structure of DNA to form transcription bubbles about 12 to 20 base pair in length. • 3 types of proteins regulate transcription initiation by RNA polymerase: Repressors Specificity factors Activators • Pyrophosphate is produced in RNA synthesis • Removal of pyrophosphates makes RNA irreversible. Removal is by phosphatase enzyme Initiation

16. RNA polymerase: • It is extracted from E.coli. It contains 4 types of polynucleotide chains α, β, β' and σ.  • The complete RNA polymerase enzyme is termed holoenzyme (α2, β, β‘, ω, σ) and other enzyme is core enzyme (α2, β, β'). • σ factor plays important role for recognizing the initiation site of transcription. After RNA synthesis, σ factor dissociates and becomes available for another transcription process several σ factor are found but most often involved in transcription is σ70.

17. The function of α2 is promoter binding, β is nucleotide binding, β' is DNA template binding and the function of ω is still unknown. • A region to which RNA polymerase binds with the aid of the σ factor is called promoter. The prokaryotic promoter sequence is not transcribed.

18. A σ base sequence (usually TTGACA) approximately 35 base pairs before the transcription starting point is present in Ecoil promoters. • TATAATG sequence or Pribnow box lies within promoter at 10 base pair before starting point of transcription or around 16 to 18 base pair from the TTGACA sequence. The RNA polymerase recognizes these sequences, binds to promoter and unwinds a short segment of DNA beginning around the pribnow box.

21. Transcription starts 6 or 7 base pairs away from 3' end of the promoter. • RNA polymerase remains at the promoter while it constructs a chain about 9 nucleotides long, then it begins to move down the template strand. • The first based used in RNA synthesis is usually a purineATP or GTP Elongation

22. Since these phosphate are not removed during transcription, the 5' end of prokaryotic mRNA has a triphosphate attached to the ribose. These triphosphate group is called Pyrophosphate. After complete RNA synthesis ,removal of pyrophosphate makes RNA irreversible and it is caused by pyrophosphatase enzyme. • Although only one polymerase can occupy the promoter at a time, more than one RNA polymerase can transcribe the same gene simultaneously.

24. Stop signals present to mark the end of a gene sequences & stop transcription by the RNA polymerase. • Prokaryotic terminators often contain a sequence coding for an RNA stretch that can form hydrogen bond to form a hairpin-shaped loop & stem structure. This structure cause RNA polymerase to pause of stop transcribing DNA. Termination

25. 1st type terminator have six uridine residues following the hairpin of mRNA & cause the polymerase to stop transcription & release mRNA without the aid of any accessory factors. • 2nd type terminator require accessory factors called rho factor as they lack poly-U residue. factor binds to mRNA & move along the molecule until it reaches RNA polymerase that has halted at a terminator. The factor then causes the polymerase to dissociate from the mRNA, probably by unwinding the mRNA-DNA complex.

28. rRNAs and tRNAs also undergo processing: Post-transcriptional processing is not limited to mRNA. rRNA and tRNA are also made from longer precursors. (1) rRNA: In bacteria 16S,23S and 5S rRNAs and some tRNAs arise from single 30S RNA precursors. 16S and 23S rRNA has modified nucleosides with the action of methylated nucleoside and pseudouridine. Cleavage occurs due to RNase(different types of RNase) Finally nucleases liberates final 16S,23S,5S rRNA and tRNA.

30. (2) tRNA: Derived from longer RNA precursors by enzymatic removal of nucleotides from 5’ and 3’ ends. Rnase P(endonuclease) removes RNA at the 5’end of tRNA. Similarly, Rnase D(exonuclease) processed at 3’ end of tRNA. Further processing is done at 3’-terminal with addition of 3’-terminal trinucleotide CCA to which amino acids is attached during protein synthesis.

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