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  2. 2. Definition Synthesis of RNA using ssDNA as a template by DNA dependent RNA polymerase • Similar to replication in terms of chemical mechanism, polarity, and use of template but differ in -does not require primers -only a short segment of DNA is transcribed
  3. 3. • All 3 types of cellular RNA’s are copied during transcription • Messenger RNAs (mRNAs) encode the amino acid sequence of one or more polypeptides specified by a gene or set of genes. • Transfer RNAs (tRNAs) read the information encoded in the mRNA and transfer the appropriate amino acid to a growing polypeptide chain during protein synthesis. • Ribosomal RNAs (rRNAs) are constituents of ribosomes, the intricate cellular machines that synthesize proteins.
  4. 4. Basic Requirements of Transcription-- prokaryotic  Template ------ssDNA  Enzyme-----RNA polymerase  Regulatory proteins  Ribonucleoside triphosphates ( A, G,C,U )  Energy –uses the energy released from the cleavage of pyrophosphates to two phosphates by pyrophosphotase
  5. 5. Steps involved in Transcription  Initiation -recognition of specific DNA sequence ( promoter region ) -beginning of bond formation process Elongation Termination & release
  6. 6. Initiation Starts with the recognition of promoter sequence on the DNA coding ( anti-template ) strand by RNA polymerase Promoter sequence • Present on the DNA  Two common sequences are present on the upstream ( 5’ ) side of the start site  Start site is denoted by +1
  7. 7. Contd--  -10 sequence & -35 sequence  5’------TTGACA--------TATAAT--------start site --------terminator 3’ (-35 ) ( -10) +1  Sequences are 6 bp long  Distance b/n these two promoters is conserved & is a separation of 17-19 nucleotides in normal  -10 sequence is called Pribnow box ( TATA box )
  8. 8. Contd—  Genes with strong promoters cause frequent initiation of transcription as often as every two seconds ( E coli ) (strong promoters have sequence that correspond closely to the consensus sequence )  Genes with weak promoters are transcribed about once in 10 minute ( weak promotes tend to have multiple substitution at that site )
  9. 9. RNA Polymerase ( E.Coli ) • Multisubunit enzyme • DNA dependent RNA polymerase • Very large molecule ( 500 kd ) and complex enzyme consisting of four kinds of subunits • Prokaryotes have single RNA pol that transcribes all the three RNA’s ( mRNA, t RNA, r RNA ) • α2 β β’ Ω σ ------holoenzyme • RNA polymerase with out σ subunit is called core enzyme • Core enzyme contains the catalytic activity.
  10. 10. Subunit Gene Number Mass ( kd ) Role α rpo A 2 37 Binds regulatory proteins β rpo B 1 151 Forms phosphodieter bond β’ rpo C 1 155 Binds DNA template σ rpo D 1 70 Recognizes promoter & initiates synthesis
  11. 11. Contd-- • RNA polymerases lack a separate proofreading 3’ to 5’ exonuclease active site (such as that of many DNA polymerases) • The error rate for transcription is higher than that for chromosomal DNA replication approximately one error for every 10 4 to 10 5 ribonucleotides incorporated into RNA.
  12. 12. Contd--  After recognizing & binding to the promoter region RNA pol synthesizes complimentary RNA sequence to the DNA template strand ( U instead T is paired with A )  RNA synthezised from 5’ to 3’ ( template read from 3’ to 5’ )  5’ end of new RNA chain is highly distinctive : a molecule starts with either pppG or pppA ( DNA synthesis – Primers )  Template strand is determined by the location of the promoter region for that gene
  13. 13. Contd--  RNA pol interacts with activator & repressor proteins that modulate the rate of transcription  In bacteria one species of RNA pol can synthezise all the RNA molecules ( mRNA, tRNA, rRNA ) except short RNA primers needed for DNA replication ( primase )
  14. 14. Elongation • RNA polymerase elongates an RNA strand by adding ribonucleotide units to the 3-hydroxyl end • The 3-hydroxyl group acts as nucleophile, attacking the phosphate of the incoming ribonucleoside triphosphate and releasing pyrophosphate. • By the time 10 nucleotides are added ,the σ factor dissociates & the core enzyme continues the elongation of the transcript
  15. 15. Contd— • The template DNA strand is copied in the 3’ to 5’ direction (antiparallel to the new RNA strand), just as in DNA replication. • Each nucleotide in the newly formed RNA is selected by Watson-Crick base-pairing interaction RNA pol (NMP)n + NTP (NMP)n+1 + PPi RNA Lengthened RNA
  16. 16. Termination E. coli has at least two classes of termination signals: • one class relies on a protein factor called ρ (rho) • the other is ρ independent.  Most –ρ independent terminators have two distinguishing features. • The first is a region that produces an RNA transcript with self complementary sequences, permitting the formation of a hairpin structure centered 15 to 20 nucleotides before the projected end of the RNA strand.
  17. 17. Contd-- • The second feature is a highly conserved string of A residues in the template strand that are transcribed into U residues near the 3 end of the hairpin. • When a polymerase arrives at a termination site with this structure, it pauses • Formation of the hairpin structure in the RNA disrupts several AUU base pairs in the RNA-DNA hybrid segment and may disrupt important interactions
  18. 18. Contd—  Rho (ρ ) dependent termination • Rho factor is an ATP dependent RNA-DNA helicases • Recognizes and bind to the termination signals and disrupts the nascent RNA/DNA complex
  19. 19. Transcription Is Regulated at Several Levels • Requirements for any gene product vary with cellular conditions or developmental stage, and transcription of each gene is carefully regulated to form gene products only in the proportions needed. • Regulation can occur at any step in transcription, including initiation ,elongation and termination. • Much of the regulation is directed at the polymerase binding and transcription initiation
  20. 20. • The binding of proteins to sequences both near to and distant from the promoter can also affect levels of gene expression. • Protein binding can activate transcription by facilitating either RNA polymerase binding or steps further along in the initiation process, or it can repress transcription by blocking the activity of the polymerase. • In E. coli, one protein that activates transcription is the cAMP receptor protein (CRP), which increases the transcription of genes coding for enzymes that metabolize sugars other than glucose when cells are grown in the absence of glucose.
  21. 21. Contd-- • Repressors are proteins that block the synthesis of RNA at specific genes. • In the case of the Lac repressor , transcription of the genes for the enzymes of lactose metabolism is blocked when lactose is unavailable
  22. 22. Inhibitors • Rifampicin ( antitubercular drug) -semisynthetic derivative ( sterptomyces) -specifically inhibit initiation not by blocking the binding of RNA pol but rather by interfering with the formation of the first phosphodiester bond -Site of action is on β subunit of RNA pol -some mutants having an altered β subunit are resistant to rifampacin
  23. 23. Actinomycin D -polypeptide derived from streptomyces -binds tightly & specifically to double stranded DNA & prevents it from being an effective template for RNA synthesis -at low concentration inhibits transcription without appreciably affecting replication & protein synthesis -inhibitor of both prokaryotic & eukaryotic cell -effective therapeutic agent in the treatment of some cancers