NUCLEIC ACIDS

1. NUCLEIC ACIDS NUCLEOTIDES DNA & RNA THE GENETIC CODE PROTEIN SYNTHESIS GENETIC REARRANGEMENTS

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

3. 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

4. Transcription in Prokaryotes • E. coli RNA Polymerase: • molecular weight about 500,000 • four different types of subunits: ,  , ’, and s • the core enzyme is 2’ • the holoenzyme is 2’s • the role of the s subunit is recognition of the promoterlocus; the s subunit is released after transcription begins • of the two DNA strands, the one that serves as the template for RNA synthesis is called the templatestrand or antisense strand; the other is called the coding (or nontemplate) strand or sense strand • the holoenzyme binds to and transcribes only the template strand

5. The Basics of Transcription

6. Promoter Sequence • Simplest of organisms contain a lot of DNA that is not transcribed • RNA polymerase needs to know which strand is template strand, which part to transcribe, and where first nucleotide of gene to be transcribed is • Promoters-DNA sequence that provide direction for RNA polymerase

7. Promoter Sequence

8. Chain Initiation • First phase of transcription is initiation • Initiation begins when RNA polymerase binds to promoter and forms closed complex • After this, DNA unwinds at promoter to form open complex, which is required for chain initiation

9. Initiation and Elongation in Transcription

10. Chain Elongation • After strands separated, transcription bubble of ~17 bp moves down the DNA sequence to be transcribed • RNA polymerase catalyzes formation of phosphodiester bonds between the incorp. ribonucleotides • Topoisomerases relax supercoils in front of and behind transcription bubble

11. Chain Elongation (Cont’d)

12. Chain Termination • Two types of termination mechanisms: • intrinsic termination- controlled by specific sequences, termination sites • Termination sites characterized by two inverted repeats

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

14. Transcription Regulation in Prokaryotes • In prokaryotes, transcription regulated by: • alternative s factors • enhancers • operons • transcription attenuation • Alternative s factors • Viruses and bacteria exert control over which genes are expressed by producing different s-subunits that direct the RNA polymerase to different genes.

15. Control by Different  Subunits

16. Enhancers • Certain genes include sequences upstream of extended promoter region • These genes for ribosomal production have 3 upstream sites, Fis sites • Class of DNA sequences that do this are called enhancers • Bound by proteins called transcription factors

17. Elements of a Bacterial Promoter

18. Operon • 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 • b-Galactosidase, an inducible protein • coded for by a structural gene, lacZ • structural gene lacY codes for lactose permease • structural gene lacA codes for transacetylase • expression of these three structural genes is controlled by the regulatory gene lacI that codes for a repressor

19. 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

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

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

22. Catabolite Repression • CAP forms complex with cAMP • Complex binds at CAP site • RNA polymerase binds at available binding site, and transcription occurs

23. Basic Control Mechanisms in Gene Control • Control may be inducible or repressive, and these may be negatively or positively controlled

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

25. 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

26. 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

27. Transcription in Eukaryotes • Three RNA polymerases are known; each transcribes a different set of genes and recognizes a different set of promoters: • RNA Polymerase I- found in the nucleolus and synthesizes precursors of most rRNAs • RNA Polymerase II- found in the nucleoplasm and synthesizes mRNA precursors • RNA Polymerase III- found in the nucleoplasm and synthesizes tRNAs, other RNA molecules involved in mRNA processing and protein transport

28. RNA Polymerase II • Most studied on the polymerases • Consists of 12 subunits • RPB- RNA Polymerase B

29. How does Pol II Recognize the Correct DNA? • Four elements of the Pol II promoter allow for this phenomenon

30. Initiation of Transcription • Any protein regulator of transcription that is not itself a subunit of Pol II is a transcription factor • Initiation begins by forming the preinitiation complex • Transcription control is based here

31. General Transcription Initiation Factors

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

33. Elongation and Termination • Elongation is controlled by: • pause sites, where RNA Pol will hesitate • anti-termination, which proceeds past the normal termination point • positive transcription elongation factor (P-TEF) and negative transcription elongation factor (N-TEF) • Termination • begins by stopping RNA Pol; the eukaryotic consensus sequence for termination is AAUAAA

34. Gene Regulation • Enhancers and silencers- regulatory sequences that augment or diminish transcription, respectively • DNA looping brings enhancers into contact with transcription factors and polymerase

35. Eukaryotic Gene Regulation • Response elements are enhancers that respond to certain metabolic factors • heat shock element (HSE) • glucocorticoid response element (GRE) • metal response element (MRE) • cyclic-AMP response element (CRE) • Response elements all bind proteins (transcription factors) that are produced under certain cell conditions

36. Response Elements

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

38. Structural Motifs in DNA-Binding Proteins • Most proteins that activate or inhibit RNA Pol II have two functional domains: • DNA-binding domain • transcription-activation domain • DNA-Binding domains have domains that are either: • Helix-Turn-Helix (HTH) • Zinc fingers • Basic-region leucine zipper

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

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

41. 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

42. Helical Wheel Structure of Leucine Zipper

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

44. Post Transcriptional RNA Modification • tRNA, rRNA, and mRNA 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 mRNA • Modifications • trimming of leader and trailer sequences • addition of terminal sequences (after transcription) • modification of the structure of specific bases (particularly in tRNA)

45. Posttranscriptional Modification of tRNA Precursor

46. Modification of tRNA • Transfer RNA- the precursor of several tRNAs is can be transcribed as one long polynucleotide sequence • trimming, addition of terminal sequences, and base modification all take place • methylation and substitution of sulfur for oxygen are the two most usual types of base modification

47. Modification of rRNA • Ribosomal RNA • processing of rRNA is primarily a matter of methylation and trimming to the proper size • in prokaryotes, 3 rRNAs in one intact ribosome • in Eukaryotes, ribosomes have 80s, 60s, and 40s subunits • base modification in both prokaryotes and eukaryotes is primarily by methylation

48. 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)

49. 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

50. Organization of Split Genes in Eukaryotes