Chapter Eleven Transcription of the Genetic Code: The Biosynthesis of RNA

1. Chapter ElevenTranscription of the Genetic Code: The Biosynthesis of RNA

2. Transcription • Overview of Transcription • synthesized on a DNA template, catalyzed by DNA-dependent RNA polymerase • ATP, GTP, CTP, and UTP are required, as is Mg2+ • no RNA primer is required • the RNA chain is synthesized in the 5’ -> 3’ direction; the nucleotide at the 5’ end of the chain retains its triphosphate (ppp) group • the DNA base sequence contains signals for initiation and termination of RNA synthesis; the enzyme binds to and moves along the DNA template in the 3’ -> 5’ direction • the DNA template is unchanged

3. Transcription in Prokaryotes • E. coli RNA Polymerase: • molecular weight about ______________________ • four different types of subunits: _____________________ • the _____________ enzyme is 2’ • the _____________ is 2’s • the role of the s subunit is recognition of the ______________; the s subunit is released after ________________ • of the two DNA strands, the one that serves as the template for RNA synthesis is called the templatestrand or ____________ strand; the other is called the coding (or nontemplate) strand or _____________ strand • the __________ binds to and transcribes only the ___________

4. The Basics of Transcription

5. Promoter Sequence • Simplest of organisms contain a lot of DNA that is __________________________ • RNA polymerase needs to know which strand is _____________strand, which part to _____________, and where first _____________ of gene to be transcribed is • _____________- DNA sequence that provide direction for RNA polymerase

6. Promoter Sequence

7. Chain Initiation • First phase of transcription is _____________ • _____________begins when RNA polymerase binds to _____________and forms ________________ complex • After this, DNA unwinds at _____________ to form open complex, required for chain ____________

8. Initiation and Elongation in Transcription

9. Chain Elongation • After strands separated, transcription _____________ of ~17 bp moves down the DNA sequence to be transcribed • RNA polymerase catalyzes formation of _____________bonds between the incorporated _____________ • Topoisomerases_____________ _____________ in front of & behind __________ bubble __________

10. Chain Elongation (Cont’d)

11. Chain Termination • Two types of termination mechanisms: 1. intrinsic termination- controlled by specific sequences called _____________ _____________, characterized by two _____________ _____________

12. Chain Termination (Cont’d) 2. Other type of termination involves rho () protein -dependent termination sequences cause hairpin loop to form

13. Transcription Regulation in Prokaryotes • In prokaryotes, transcription regulated by: __________________________________ __________________________________ __________________________________ __________________________________ • Alternative s factors • ___________ & ____________ exert control over which genes are expressed by producing different s-subunits that direct the RNA _____________ to different genes.

14. Control by Different  Subunits

15. Enhancers • Certain genes include sequences upstream of ____________________________________ • These genes for ribosomal production have _____ upstream sites, _________ sites • Class of DNA sequences that do this are called ______________ • Bound by proteins called _________ _________

16. Elements of a Bacterial Promoter

17. Operon • _________ : a group of operator, promoter, and structural genes that codes for proteins • the control sites, promoter, and operator genes are physically adjacent to the structural gene in the DNA • the regulatory gene can be quite far from the operon • operons are usually not transcribed all the time • __________________, an inducible protein • coded for by a structural gene, _________ • structural gene _________ codes for lactose permease • structural gene _________ codes for transacetylase • expression of these 3 structural genes is controlled by the regulatory gene ______that codes for a repressor

18. How Does Repression Work • Repressor protein made by lacI gene forms tetramer when it is translated • Repressor protein then binds to operator portion of operon • Operator and promoter together are the control sites

19. Binding Sites On the lac operon • Lac operon is induced when E. coli has _________ as the carbon source • Lac protein synthesis repressed by ____________ (catabolite repression) • E. coli recognizes presence of glucose by promoter as it has 2 regions: RNA polymerase binding site, _________ _________ _________ (CAP) binding site

20. Binding Sites On lac operon (Cont’d)

21. Catabolite Repression • CAP forms complex with _________ • Complex binds at CAP site • RNA polymerase binds at available binding site, and _____________ occurs

22. Basic Control Mechanisms in Gene Control Control may be _________ or _________, and these may be _________ or _________ controlled

23. Control of the trp operon • Trp operon codes for a leader sequence (trpL) & 5 polypeptides • The 5 proteins make up 4 different enzymes that catalyze the multistep process that converts chorisimate to tryptophan

24. Alternative 2˚ structures can form in trp operon • These structures can form in the leader sequence • Pause structure- binding between regions 1 and 2 • Terminator loop- binding between regions 3 and 4 • Antiterminator structure- Alternative binding between regions 2 and 3

25. Attenuation in the trp operon • Pause structure forms when ribosome passes over Trp codons when Trp levels are high • Ribosome stalls at the Trp codon when trp levels are low and antiterminator loop forms

26. Transcription in Eukaryotes • Three RNA polymerases are known; each transcribes a different set of genes and recognizes a _____________________________________: • RNA Polymerase I- found in the _________ and synthesizes precursors of most _________ • RNA Polymerase II- found in the _________ and synthesizes _________ precursors • RNA Polymerase III- found in the _________ and synthesizes _________, other RNA molecules involved in mRNA _________ and _________ _________

27. RNA Polymerase II • Most studied of the polymerases • Consists of 12 subunits • ______ - RNA Polymerase B

28. How does Pol II Recognize the Correct DNA? 4 elements of the Pol II promoter allow for this phenomenon

29. Initiation of Transcription • Any protein regulator of transcription that is not itself a subunit of Pol II is a _________ _________ • Initiation begins by forming a _________ _________ • Transcription control is based here

30. General Transcription Initiation Factors

31. Transcription Order of Events • Less is known about _________ than _________ • The phosphorylated Pol II synthesizes RNA and leaves the promoter region behind • GTFs are left at the promoter or dissociate from Pol II

32. Elongation and Termination • Elongation is controlled by: • _________ sites, where RNA Pol will hesitate • _________, which proceeds past the normal termination point • _________ _________ _________ _________ (P-TEF) and _________ _________ _________ _________ (N-TEF) • Termination • begins by stopping RNA Pol; the _____________ consensus sequence for termination is ___________

33. Gene Regulation • _________ & _________ - regulatory sequences that augment or diminish transcription, respectively • DNA _________ brings _________ into contact with transcription _________ and ____________

34. Eukaryotic Gene Regulation • _________ _________ are enhancers that respond to certain metabolic factors • _________ _________ _________(HSE) • _________ _________ _________(GRE) • _________ _________ _________(MRE) • _________ _________ _________(CRE) • Response elements all bind _________(transcription factors) that are produced under certain cell conditions

35. Response Elements

36. Activation of transcription Via CREB and CBP • _________________ CREB does not bind to CREB binding protein, and no transcription occurs • _________________ of CREB causes binding of CREB to CBP • Complex with basal complex (RNA polymerase and GTFs) activates _________________

37. Structural Motifs in DNA-Binding Proteins • Most proteins that activate or inhibit RNA Pol II have two _________ _________ : • DNA-binding domain • transcription-activation domain • DNA-Binding domains have domains that are either: • _________________________ • _________________________ • _________________________

38. Helix-Turn-Helix Motif Hydrogen bonding between amino acids and DNA

39. Zinc Finger Motif • Motif contains 2 cysteines and 2 His --12 amino acids later • Zn binds to the repeats

40. Basic Region Leucine Zipper Motif • Many transcription factors contain this motif, such as CREB (Biochemical Connections, page 315) • Half of the protein composed of basic region of conserved Lys, Arg, and His • Half contains series of Leu • Leu line up on one side, forming hydrophobic pocket

41. Helical Wheel Structure of Leucine Zipper

42. Transcription Activation Domains • _________________________ domains - rich in Asp and Glu. Gal4 has domain of 49 amino acids, 11 are acidic • _________________________ domains - Seen in several transcription factors. Sp1 has 2 glutamine-rich domains, one with 39 Glu in 143 amino acids • _________________________ domains - Seen in CTF-1 (an activator). It has 84 amino acid domain, of which 19 are Pro

43. Post Transcriptional RNA Modification • ____________________________________ are all modified after transcription to give the functional form • the initial size of the RNA transcript is greater than the final size because of the leader sequences at the 5’ end and the trailer sequences at the 3’ end • the types of processing in prokaryotes can differ greatly from that in eukaryotes, especially for _______________ • Modifications • ____________________ of leader and trailer sequences • addition of _____________ _________ (after transcription) • modification of the structure of specific bases (particularly in _______________)

44. Posttranscriptional Modification of tRNA Precursor

45. Modification of tRNA • tRNA- the precursor of several tRNAs is can be transcribed as ________ polynucleotide sequence • ____________________________________________________________ all take place • ____________________ & ________________ of ____________________ are the two most usual types of base modification

46. Modification of rRNA • Ribosomal RNA • processing of rRNA is primarily a matter of ________________________________________ • in _______________, 3 rRNAs in one intact ribosome • in _______________, ribosomes have 80s, 60s, and 40s subunits • base modification in both prokaryotes and eukaryotes is primarily by _____________________

47. Modification of mRNA • Includes the capping of the 5’ end with an N-methylated guanine that is bonded to the next residue by a 5’→5’ triphosphate. • Also, 2’-O-methylation of terminal ribose(s)

48. mRNA Modification • A polyadenylate “tail” that is usually100-200 nucleotides long, is added to the 3’ end before the mRNA leaves the nucleus • This tail protects the mRNA from nucleases and phosphatases • Eukaryote genes frequently contain intervening base sequences that do not appear in the final mRNA of that gene product • Expressed DNA sequences are called exons • Intervening DNA sequences that are not expressed are called introns • These genes are often referred to as split genes

49. Organization of Split Genes in Eukaryotes

50. The Splicing Reaction • Exons are separated by intervening _______________ • When the exons are spliced together, a _______________ forms in the intron