Chapter 3. DNA, RNA, and Protein Synthesis

Transcription

1 Chapter 3. DNA, RNA, and Protein Synthesis

2 4. Transcription

3 Gene Expression Regulatory region (promoter) 5 flanking region Upstream region Coding region 3 flanking region Downstream region Transcription Factor RNA Polymerase Gene (ORF) Terminator DNA Transcription 5 UTR 3 UTR mrna Translation Protein

4 RNA Structure Ribose: both 2 and 3 OH Uracil(U) instead of thymine (T) Mostly single strand Secondary structure Intrastrand base-pairing Composition of RNA in metabolically active cell mrna: 3~5 % rrna: 90% trna: 4%

5 Types of RNA

6 RNA Polymerase RNA synthesis using ssdna as a template Prokaryote One RNA polymerase Eukaryote RNA Pol I rrna (28S, 5.8S, 18S) RNA Pol II mrna RNA Pol III trna, 5S rrna 5 to 3 RNA synthesis

7 RNA Polymerase

8 Genes Gene Specific nucleotide sequence that is transcribed into RNA Structural gene Gene encoding protein Polycystronic transcription Prokaryote only Transcription of multiple genes in one transcription unit Operator promoter OFR1 OFR2 OFR3 Terminator mrna

9 mrna Structure

10 Genes Can Be Transcribed with Different Efficiencies

11 Three Phases of Transcription Initiation :Binding of RNA polymerase to template DNA Elongation : Synthesis of RNA (5 to 3, NTP) Termination : Release of the enzyme and RNA from the DNA template

12 Prokaryotic RNA polymerase Holoenzyme a 2 bb w (Core enzyme) Catalytic subunits for RNA polymerization s Determine promoter specificity

13 Transcription Initiation in Prokaryotes s a 2 bbw +1 TTGACA TATAAT Gene (ORF) -35 Box -10 Box 20 to 600 nt

14 Transcription Initiation in Prokaryotes Closed Complex a 2 bb s 5 TTGACA TATAAT 3 3 AACTGT ATATTA s a 2 bb Open Complex TTGACA TAT 3 3 AACTGT ATA 5 Coding, Sense Anticoding, Antisense Template pppa or pppg

15 Transcription Cycle of Prokaryotic RNA polymerase

16 Directions of Transcription

17 Transcription Elongation and Termination DNA unwinding: ~17 bp ~12 bp

18 Transcription Termination in Prokaryotes (1) ρ(rho) -independent terminator Region of dyad symmetry centered about bases upstream of the last nucleotide in the transcript. Absolute sequence is not conserved, but the hairpin structure is conserved. A string of ~6 U's in RNA product 5 UUUUUU

19 Model for the mechanism of rho-independent termination 1. Formation of the hairpin causes RNA polymerase to pause. 2. Formation of the hairpin in the nascent RNA limits the RNA/DNA hybrid to a short segment of da/u base pairs 3. Nascent RNA is released, DNA/DNA hybrid re-forms, and RNA polymerase falls off

20 Transcription Termination in Prokaryotes (2) ρ(rho) -dependent terminator Hairpin structure similar to rho-independent terminators Lack the poly-u stretch Require the activity of a rho-protein, a trans-acting factor that functions as a homohexamer

21 Model for the mechanism of rho-dependent termination 1. Rho translocates 5' to 3' along nascent RNA 2. Rho binds consensus sequence (rut : rho utilization) located approximately 100 bases upstream of the transcriptional termination site 3. RNA polymerase pauses at hairpin 4. Rho unwinds DNA/RNA hybrid (RNA-dependent ATPase activity)

22 Regulation of Transcription Initiation in Prokaryotes Different s factors Recognize different consensus sequences e.g. E. coli s 70 s 54 s 32 s s Transcription factors Activators Repressors Negative supercoiling Gyrase (Topoisomerase II) generates negative supercoiling Topoisomerase I Relaxes excess negative supercoiling

23 Regulation of mrna Transcription in Prokaryotes Negative controlled system Repressor Repression in the presence of effector molecule Repression with a coreporessor

24 Trp Repressor

25 Regulation of mrna Transcription in Prokaryotes Positive controlled system Activator Promoting RNA polymerase activity Activated or repressed by effectors

26 Repressors and Activators

27 5. Transcription in Eukaryotes

28 Components of Transcriptional Control in Eukaryotes Cis elements: Regulatory DNA sequences Promoter RNA polymerase binding site (TATA box) Promoter-proximal elements Enhancer (eukaryotes only) Trans elements: Specific proteins binding to the regulatory sequence RNA polymerase complex RNA polymerase General transcription factors Mediators Transcription factors Activators Repressors

29 Promoter Elements in Eukaryotic Transcription TATA box: direct RNA polymerase bp upstream of TC start site Promoter proximal elements: 10 to 20 bp < 200 bp of TC start site Enhancer: Composed of multiple elements of bp, 100 bp long Upto 50 kb upstream or downstream, or within an intron

30 RNA Polymerase: E.coli vs. Yeast

31 RNA Polymerase in Eukaryotes Polymerase Genes transcribed RNA Pol I Ribosomal RNA genes (28S, 5.8S, 18S) RNA Pol II RNA Pol III 40S 60S 18S rrna 30 different proteins 28S, 5.8S, and 5S rrna. 45 different proteins Protein-coding genes Most small nuclear RNA (sn RNA) genes Genes for trna 5S ribosomal RNA U6-sn RNA small nucleolar (sno) RNA Ribosome

32 Pre-initiation complex (PIC) of RNA Pol II RNA Polymerase II 2 Large subunits 10 small subunits General transcription factors (TFIIA), TFIIB, TFIID, TFIIE, TFIIF, and TFIIH TBP (TATA-box binding protein) TAFs (TBP associated factors)

33 Assembly of PIC TBP

34 Regulation of Transcription initiation Mediator Mediate activated transcription by binding to transcription factors and RNA Pol II SWI/SNF Chromatin remodeling SAGA Histone acetyltransferase (HAT) complex

35 Chromatin Remodeling and Histone Modification

36 Transcription Initiaion

37 Ordered Assembly of Transcription Initiation Complex

38 Molecular Mechanisms of Transcription Activation and Repression Modulation of Chromatin structure Chromatin remodeling Acetylation and deacetylation: Acetylation of Histone H3, H4 Unstructured chromatin Facilitate assembly of transcription initiation complex Heterochromatin: Condensed chromain, telomeres, centromeres Euchromatin: most transcribed genes are located Interaction with Pol II and general transcription factors

39 Overview of Transcription Control in Multicellular Eukaryotes

40 Mechanism of Histone Deacetylation in Yeast Transcriptional Control

41 Mechanism of Histone Acetylation in Yeast Transcriptional Control

42 Exon: 150~ 200 bp Intron: 40 ~ 10,000 bp Eukaryotic Structural Gene Promotor Inr Exon Intron Exon t Transcription Primary mrna 7 mg AAAAA Splicing mrna 7 mg AAAAA Translation

43 Processing of mrna 5 Capping Splicing 3 Polyadenylation

44 5 Capping 5 end blocked by the addition of a 7- methylguanosine. The cap structure has no free phosphates Protected from attack by phosphatases, or nucleases

45 Polyadenylation The polyadenylation sequence is bound by a cleavage and polyadenylation specificity factor (CPSF). An endonuclease cleaves the transcript. PolyA polymerase binds to CPSF, adding up to 250 adenylate residues to the 3' end of the transcript.

46 Consensus Sequence for Splicing in Humans

47 Pre mrna Splicing

48 Mechanisms Splicing by Spliceosome

49 Alternative Splicing Different pattern of exon splicing from a primary transcript Generation of tissue-specific proteins from the same structural gene

50 The Export of mrna Through the Nuclear Pore