Modified Central Dogma of Francis Crick (1958)

1. Gene ExpressionProkaryotic Gene TranscriptionG & G Chapter 299/14/11Thomas Ryan, Ph.D. Biochemistry and Molecular Geneticstryan@uab.edu

2. Modified Central Dogma of Francis Crick (1958)

3. Prokaryotic Chromosome (E. coli) • Large circular chromosome - 4.6 x 106 bp • Genome forms a compact structure called the nucleoid • DNA organized in 50-100 loops (domains) • The ends of loops are constrained by binding to protein structure which is in contact with cell membrane

4. Additional Forms of RNA All cells transcribe 3 major types of RNA molecules: messenger RNA (mRNA) ribosomal RNA (rRNA) transfer RNA (tRNA) In archea and eubacteria: 3 RNAs are produced by a single DNA dependent RNA polymerase In eukaryotes: 3 RNAs are produced by 3 distinct DNA dependent RNA polymerases (I, II, & III)

5. RNA Quantity and Stability • rRNA & tRNA (stable): • Not degraded rapidly (although extensively processed) • Rapidly growly E. coli: • 80% of RNA is rRNA • 15% is tRNA • Ribosome number [rRNA] is proportional to growth rate • mRNA (translated): • ~2 to 5% of total RNA is mRNA • unstable with a t1/2 ≈ 2-3 min - allows regulation at the level of mRNA synthesis

6. Exonucleases (3’—> 5’ only in bacteria) 5’ 3’ Endonucleases (internal cuts) 5’ 3’ mRNA Degradation by RNases

7. Transcription: The Players Ribonucleotides (NTPs) Template (DNA) DNA Dependent RNA Polymerase Transcription factors

8. Transcription: DNA to mRNA G & G page 907

9. Major bases found in DNA and RNA DNARNA Adenine Adenine Cytosine Cytosine Guanine Guanine Thymine Uracil (U) thymine-adenine base pair uracil-adenine base pair

10. A A T T C C G G DNA Dependent RNA Polymerase: Catalysis Reaction Growing RNA strand Template strand DNA • Direction of synthesis is 5’ to 3’ • No primer required • Template required n n+1

11. Gene [ ] 5’ AGTC 3’ DNA 3’ TCAG 5’ Transcription RNA AGUC 5’ 3’ * Chromosome is divided into genes, which encode RNA and protein products. Chemical differences ribosevs. deoxyribose *uracilvs. thymine • single stranded • complementary to one strand of DNA (bottom in this case)

12. Naming the DNA Strands of a Gene nontemplate nontranscribed top [ ] 5’ AGTC 3’ DNA 3’ TCAG 5’ bottom Transcription template transcribed RNA AGUC 5’ 3’

13. Both DNA Strands Encode Genes And Can Be Transcribed. RNA # 2 3’ 5’ Transcription [ [ ] ] 5’ 3’ P Gene # 1 Gene # 2 DNA P 3’ 5’ Transcription 5’ 3’ RNA # 1

14. Prokaryotic RNA polymerase • Synthesizes all major classes of RNA • messenger RNA (mRNA) • ribosomal RNA (rRNA) • transfer RNA (tRNA) • Multisubunit Protein • Holoenzyme = a2bb' catalyzes initiation of RNA synthesis specifically at a promoter • Core enzyme= a2bb' catalyzes elongation of the RNA chain

15. Transcription in Prokaryotes Only a single RNA polymerase • In E.coli, RNA polymerase is 465 kD complex, with 2 , 1 , 1 ', 1  •  subunits appear to be essential for assembly and for activation of enzyme by regulatory proteins •  binds NTPs, interacts with , and forms catalytic site with b' • ' binds nonspecifically to DNA and forms catalytic site with b •  recognizes promoter sequences on DNA, aids in melting the dsDNA by binding nontemplate strand

16. Prokaryotic Transcription Cycle • Initiation • Holoenzyme binds to the promoter, unwinds DNA, and forms phosphodiester bonds between 7 to 12 nucleotides • Need s to recognize promoter • Elongation • s dissociates • Core enzyme elongates RNA with high processivity • Termination • Polymerase dissociates from template DNA and releases new RNA • Rho()-factor dependent or independent.

17. Binding of RNA Polymerase to Template DNA • Polymerase binds nonspecifically to DNA with low affinity and migrates, looking for promoter • Sigma (s) subunit recognizes promoter sequence • RNA polymerase holoenzyme and promoter form "closed promoter complex" (DNA not unwound) - Kd = 10-6 to 10-9 M • Polymerase unwinds about 14 base pairs of DNA to form "open promoter complex" - Kd = 10-14 M

18. RNA Polymerase Binding to DNA - Promoter Search Nonspecific binding to DNA: holo - Ka ≈ 107/M (i.e., to non-promoter DNA) (very rough numbers) Specific binding to DNA: holo - Ka ≈ 1014/M (i.e., to promoter) (actual value depends on promoter!) Note: in E. coli there are: ~3000 molecules of RNAP core ~1000 molecules of s ~1000 promoters virtually unlimited nonspecific DNA sites • Holoenzyme searches for promoters by sliding along DNA and by intramolecular transfer on the chromosome.

19. Properties of Promoters See Figure 29.3 • Promoters typically consist of 40 bp region on the 5'-side of the transcription start site • Two consensus sequence elements: • The "-35 region", with consensus TTGACA • The Pribnow box near -10, with consensus TATAAT - this region is ideal for unwinding - why?

20. Prokaryotic Promoters G & G Fig. 29.3

21. Consensus s Factor Promoters

22. Stages of Transcription See G & G Figure 29.2 • binding of RNA polymerase holoenzyme at promoter sites • initiation of polymerization • chain elongation • chain termination

23. Transcriptional Events

24. Initiation of Polymerization • RNA polymerase has two binding sites for NTPs • Initiation site prefers to binds ATP and GTP (most RNAs begin with a purine at 5'-end) • Elongation site binds the second incoming NTP • 3'-OH of first attacks alpha-P of second to form a new phosphoester bond (eliminating PPi) • When 7-12 unit oligonucleotide has been made, sigma subunit dissociates, completing "initiation" • Note mode of action of rifamycin (rifampicin)--binds to b subunit of RNA polymerase and blocks first phosphodiester bond. Specific for prokaryotic RNA polymerase!

25. Events at initiation of transcription

26. Chain Elongation Core polymerase - no sigma • Polymerase is accurate - only about 1 error in 10,000 bases • Even this error rate is OK, since many transcripts are made from each gene • Elongation rate is 20-50 bases per second - slower in G/C-rich regions (why??) and faster elsewhere • Topoisomerases precede and follow polymerase to relieve supercoiling

27. The Elongation Complex • RNAP core enzyme covers about 60 bp of DNA, with about 17 bp unwound = transcription bubble. • The bubble must contact the active site for polymerization. • At the beginning of the bubble, the DNA is unwound, implicating a helicase activity. • At the end of the bubble, the DNA is rewound.

28. Supercoiling Versus Transcription G & G Fig. 29.4

29. Inhibitors of Transcription Intercalates G:C basepairs b subunit of RNAP

30. Transcription Termination Two mechanisms • Rho - the termination factor protein • rho is an ATP-dependent helicase • it moves along RNA transcript, finds the "bubble", unwinds it and releases RNA chain • Specific sequences - termination sites in DNA • inverted repeat, rich in G:C, which forms a stem-loop in RNA transcript • 6-8 As in DNA coding for Us in transcript • “Intrinsic Termination”

31. Transcription Termination Sequence Dependent / Factor Independent (Intrinsic Termination)

32. Stem Loop Structure

33. Transcription Termination: Rho-factor Dependent

34. Regulation of Prokaryotic Gene Transcription Regulation occurs at every level Transcription (RNA synthesis) RNA stability, processing, localization Translation Post-translational

35. Regulation of Prokaryotic Gene TranscriptionIntroduction • DNA:protein, protein:protein interactions • Organization of genes into operons • lac and trp operons • Positive control or activation • Negative control or repression • Attenuation control of transcription

36. Transcription Terminology • promoter = DNA site recognized by RNA polymerase for specific transcriptional initiation • terminator = region of DNA containing signals for termination of transcription • structural gene = DNA that encodes a protein (or RNA product?) • cistron = gene, mRNA specified by the structural gene • coding region = structural gene or cistron • open reading frame (ORF) = coding region (i.e., no stop codons) • operon = promoter + (gene)n + terminator, where n ≥1 1 transcript ≥ 1 cistron

37. General Rules for DNA Binding Proteins • 3D structure of regulatory proteins - most of them are homodimers • DNA sequence recognized by homodimers are typically palindromic (inverted repeats); they have dyad symmetry • Each monomer of the homodimer is in contact with bases in half of the palindromic sequence • This allows the protein coding region to remain relatively small while the protein recognizes a large sequence that is quite specific

38. Transcription factors DNA binding proteins that decrease (repressors) or increase (activators) the efficiency of transcription at the promoter.

39. Transcription is the primary site of control in prokaryotes • Promoters drive the expression of genes • Prokaryotic genes can be arranged in operons

40. Transcription Regulation in Prokaryotes • Genes encoding for enzymes of metabolic pathways are grouped in clusters on the chromosome - called operons • This allows coordinated regulation and gene expression • A regulatory sequence adjacent to such a unit determines whether it is transcribed - this is the ‘operator’ • Regulatory proteins interact with operators to control transcription of the genes

41. General Organization of Operons Operators can be upstream, downstream, or overlapping with the promoter. Regulatory proteins that bind to the operator can influence the access of RNA polymerase to the promoter thereby affecting the rate of transcription initiation.

42. Coordinate Regulation • Expression of several or numerous genes can be controlled simultaneously. • Operon: a set of genes that are transcribed from the same promoter and controlled by the same operator site and regulatory proteins. • Regulon: a set of genes (and/or operons) expressed from separate promoter sites, but controlled by the same regulatory molecule. Global regulons may coordinate expression of many genes and operons, and may induce some, but repress others.

43. Global Regulation Via Sigma Factors Different promoter architectures are recognized by sigma factor subunit of RNA polymerase

44. Gene ExpressionProkaryotic Gene Transcription(Cont.)&Eukaryotic TranscriptionHistones and ChromatinG & G Pages 336-340, Chapter 29, 9/14/11Thomas Ryan, Ph.D. Biochemistry and Molecular Geneticstryan@uab.edu