Fig. 11-CO, p.264

1. Fig. 11-CO, p.264

2. Learning Objectives • How Does Transcription Take Place in Prokaryotes? • How Is Transcription Regulated in Prokaryotes? • How Does Transcription Take Place in Eukaryotes? • How Is Transcription Regulated in Eukaryotes? • How Is RNA Modified after Transcription? • How Does RNA Act as an Enzyme?

3. Gene expression It is the action of a gene to produce single polypeptide, which occurs in two steps:  Transcription of the information encoded in single gene of chromosomal DNA into a molecule of RNA.  Translation of the information encoded in the nucleotides of mRNA into defined sequence of amino acids in a polypeptide

4. RNA transcription Transcription produces RNA molecules that are complimentary copies to one strand in DNA molecule.

5. The transcription of each gene starts from specific sequence on DNA called the promoter and finishes on another sequence called the terminator. Both the promoter and terminator are regulatory regions separated from the coding sequence of the DNA

6. Unlike DNA replication, the transcription is unidirectional and only one of the two double DNA strands act as a template. Since, the Synthesis of RNA proceeds in the 5' 3' direction, therefore in each transcription step only the DNA strand having the 3’ 5' direction is copied as a template. The copying process involves small part but not the whole DNA molecule.

7. Table 11-1, p.265

8. The basics of transcription Fig. 11-1, p.265

9. Prokaryotic RNA transcription Transcription in prokaryotes is carried out by a single RNA polymerase and performed in 3 stages: initiation, elongation and termination.

10. The promoter region contains two boxes of specific sequences located -10 bp and -35 bp upstream (left side) of position +1, which represents the first nucleotide on DNA strand to be copied as a template.

11. With the help of sigma factor binding, the RNA polymerase can strongly interact with these two promoter boxes and produce large amounts of RNA. Any binding of RNA polymerase at promoter regions other than these two specific boxes will be weak and produces little RNA.

12. The most common nucleotide sequence found in these two boxes are TATAAT at -10 region and TTGACA at -35 region

13. Sequence of representative promoters from E.coli E. coli have 7 different sigma factors B. subtilis have 18 different sigma factors Fig. 11-2, p.266

14. The binding of sigma-RNA polymerase complex at the -35 box also cause unwinding of short DNA segment extended between -10 to +1 bp sequence to make enough space for the initiation of transcription process. In this process the interacting complex undergoes a conformational shift that leads to unwinding of the double helix.

15. Elongation The open complex begins RNA synthesis complementary to one of the strands of DNA called sense strand, the opposite DNA strand is called antisense strand. At this point sigma factor dissociates from the RNA polymerase enzyme which can be recycled again for continued initiation of transcription.

16. During elongation of the RNA molecule, ribonucleoside triphosphates of the bases A, U, G, and C pair with complementary bases in the sense or template strand of DNA and are then connected with 3'–5' phosphodiester bonds by RNA polymerase. Thus, the RNA molecule grows from its 5' end toward its 3' end (5' → 3'). The DNA double helix unwinds ahead of the advancing RNA polymerase to expose more of the sense strand for extending the RNA chain. The DNA reforms its double-helical shape after the enzyme has passed a given region

17. Transcription occurs at 30-50 nucleotides/second adding ribonucleotides monophosphate (supplied as triphosphates ) in to RNA chain using the antisense strand as a template. As each nucleoside triphosphate is brought to the 3' end of the growing strand, the two terminal phosphates are removed as pyrophosphates similar to the reaction catalyzed by DNA polymerase. The addition proceeds in the 5' 3' direction and follow base pair ruling taking in consideration that each A on the DNA guides the insertion of the pyrimidine uracil (U) instead of T. The proofreading by the enzyme insures the correct insertion of bases.

18. Sequence of events in the initiation & elongation phases of transcription in prokaryotes Fig. 11-3, p.268

19. Termination In bacteria transcription termination occurs by two general mechanisms. The first mechanism is called Rho dependant. The Rho is a protein - a termination factor (similar to sigma initiation factor) which recognizes a specific DNA sequence and interacts with the transcription machinery to terminate transcription .At that site the Rho binds to separate the new RNA from Polymerase and DNA template. The second mechanism is called Rho – independent because it acts independently of Rho factor

20. The ρ-independent terminators have a dyad symmetry in the double-stranded DNA, centered about 15–20 nucleotides before the end of the RNA, and have about six adenines in the sense strand that are transcribed into uracils at the end of the RNA. dyad symmetry are two areas of a DNA strand whose base pair sequences are inverted repeats of each other such as the sequences GAATAC and GTATTC which are reverse complements of each other. The RNA transcript of the dyad symmetry folds back on itself to form a hairpin structure, ending with approximately 6 uracils .Because an RNA-DNA hybrid consisting of polyribo-U and polydeoxyribo-A is very unstable, the RNA chain is quickly released from the DNA duplex. .

21. Inverted repeats terminate transcription Fig. 11-5, p.269

23. The rho(p)factor mechanism of transcription termination Fig. 11-6, p.270

24. Eukaryotic RNA transcription Transcription in eukaryotes differs from the process in prokaryotes by the following properties: 1. Genes are transcribed individually instead of groups. 2. Transcription occurs in a separate compartment (the nucleus) from translation, which occurs in the cytoplasm 3. Initially transcription results in a pre-messenger RNA (pre-mRNA) molecule that must be processed before it emerges as a mature mRNA ready for translation 4. DNA is complexed with many proteins and is highly compacted, and therefore must be unwound to expose its promoters. Only genes occurring in regions of the relaxed chromatin (unpacked) are prepared for potential transcription .

25. Most transcriptionally inactive segment of DNA are found in the highly condensed heterochromatin. Interconversion of the two forms is called chromatin remodeling. Two major factors influence chromosome structure. They are DNA methylation and histone acetylation. Genes with more methylation are less active in transcription, while more acetylated histones produce looser chromatins, which become more active. Relaxed chromatin Highly packed chromatin Genomic sequences packaged into Genomic sequences free nucleosomes are accessible to organized into solenoid transcription configuration are inaccessible to transcription machinery

27. 5. Eukaryotes have three separate RNA polymerases instead of one , each transcribing certain type of RNA. RNA Polymerase I rRNAs (ribosomal RNA) RNA Polymerase II mRNA precursors or heterogenous RNA(hn-RNA) RNA Polymerase III tRNA (transfer RNA) and other small RNA RNA polymerase II structure is similar to the core structure of prokaryotic polymerase, but it has a larger protein size with about 8 to 14 subunits instead of 4. The largest subunit of RNA polymerase II has a carboxy-terminal domain and called CTD. This subunit can be phosphorylated by certain kinase

28. 6. Unlike prokaryotes, RNA polymerase II is unable to bind directly the promoter region because it has no similar sigma subunit of their prokaryotic . Instead, eukaryotic polymerases depend on other proteins that bind to the promoter regions and then bring the RNA polymerases to the correct promoter site.

29. Promoter region of polymerase II Unlike prokaryotic promoters, eukaryotic promoter regions do not have a fixed sequence for starting the initiation of transcription. The most common promoter sequence as initiator element is "TATA box", with the sequence TATAAAA . This region approximately 25 base pairs upstream from the starting nucleotide and almost similar to the -10 region found in prokaryotic promoters.

30. Other non-universal sequences include the "CAAT box" (GGCCAATCT) and the "GC box" (GGGCG) located in promoter regions more than 100 base pairs distance from the starting transcription point. Each of these sequences can act as binding sites for specific transcription factors to assist in the binding of RNA polymerases. However, the presence of these three boxes together is more common than their individual presence in the promoter region. TATA Box By Itself Makes for Only a “Weak” Promoter

31. • Additional promoter sequences are needed for a strong promoter OR for regulated promoters that provide binding sites for Gene-specific Transcription In eukaryotes, the specific promoter sequence for a gene transcribed by pol II is called core promoter and most often found immediately upstream (5′) of the start site for the gene

32. General transcription factors for eukaryotic RNA polymerase II • The transcription factors are proteins that control the activity of RNA pol II and because they are required for all mRNA genes they are called general transcription factors. • TFII means general transcription factor of RNA pol II

33. RNA polymerase II requires Six General TF’s: TFIIA ,TFIIB ,TFIID ,TFIIE ,TFIIF ,TFIIH • . TFIID is transcription factor D for RNA polymerase II . TFIID which is the key factor to Promoter Recognition • TFIID itself is a multi-protein complex composed of TATA box binding protein (TBP) + the protein TATA-associated factors (TAF).TBP plays a role in directing RNA polymerase to initiate at the correct place

34. Initiation of transcription: General transcription factors and the polymerase undergo a pattern of sequential binding to initiate transcription of nuclear genes. Formation of pre-initiation Complex • The TFIID interacts with the TATA box sequence of the DNA through the TBP component to form TFs (transcription factors) complex. This initial binding event creates binding sites for TFIIA and TFIIB to be formed. • TFIIF already bound to RNA pol II which can bring it to bind the TFs complex at the TATA box region.

35. Further addition of TFIIE and TFIIH completes the formation of pre- initiation complex . The transcription factors bind to DNA in a preferred order: TFIID, then TFIIA, then TFIIB, then RNA Pol II + TFIIF, then TFIIE

38. The template DNA is still a duplex and the next step in the initiation process is the unwinding of the duplex DNA at the start site for transcription. Once Preinitiation Complex Forms, TFIIH binding will activate the polymerase clearance from the promoter region to start elongation. • TFIIH has both kinase and helicase activities • The previous binding of RNA pol II to TFIID occurred through unphosphorylated C-terminal Domain (CTD) of β’-subunit in the enzyme.

39. TFIIH (kinase) phosphorylates CTD to release RNA pol II and activates the promoter clearance. Once the polymerase has begun catalyzing phosphodiester bond formation, then the complex is called an initiation complex and allows a minimal level of transcription to be initiated at a precisely defined site downstream from the TATA box • Further enhancement (or repression) of that transcription is achieved by interactions with additional transcription factors that bind to upstreampromoter elements, enhancers, and silencers.

40. Enhancers a. can be located at great distances (>1000 bps) from start site of transcription either from the 5' or 3' end of gene b. stimulates transcription (~100 times) c. transcription factors bound to enhancer will stimulate binding of RNAP II to promoter regions closer to the start site of transcription.

41. Elongation It is carried out by RNA polymerase of the initiation complex similar to the process of prokaryotic transcription.

42. Termination The termination involves recognition of certain sequence at the end of the gene without the need for extra factors like prokaryotes.

43. END PART I

44. Processing of mRNA precursor in eukaryotes Eukaryotic genes contain segments called introns, which break up the amino acid coding sequence into different segments called exons. In transcription, both exons and introns are expressed to produce preliminary large mRNA called heterogeneous RNA (hnRNA ). The hnRNA contains sequences expressed from both exons (coding regions) and introns (non coding) regions on DNA. This RNA must be processed in before it is translated into the polypeptide.

46. Processing of hnRNA The processing of hnRNA occurs in the nucleus and involves removal of the introns sequences as well as the chemical modifications of both RNA ends in order to produce the mature RNA that will be functional in translation.

47. 1. Removal of introns (RNA splicing) Splicing is carried out by small nuclear ribonucleoprotein complex(snRNPs )called the spliceosome, which contains the following components: a. Special type of RNA called small nuclear RNA b. Protein factors c. Endonuclease d. ligase enzyme.

48. The first step in RNA splicing is the formation of a loop ,stabilized by the sequence present in small nuclear RNA which is complementary to the two splicing ends of an intron.

49. The endonuclease in the spliceosome complex will make cuts at each end of the intron loop to release the intron while the free ends of the two left exons are rejoined by the complex ligase activity. The removed intron will be degraded, leaving the exons sequence to be spliced together.

50. Splicing of mRNA precursors ( a lariat forms in the intron ) Fig. 11-34, p.295