1. Prokaryotic Transcription

4. Phases of transcription

5. RNA Polymerase

6. RNA Polymerase

10. Sigma 70 binding sites Region 3.2 acts as molecular mimic in abortive initiation; region 2.3 melts DNA

11. Open complex- note regions of binding by σ 70 binds to UP-element

12. The Transcription Unit

13. Transcription Occurs in a Bubble

14. Synthesis Occurs in the 5 ´ to 3´ Direction

15. Initiation

17. Transcription Requires Gyrase and Topoisomerase

19. Intrinsic Termination

20. Rho-Dependent Termination

21. Antitermination

22. Eukaryotic Transcription

23. Regulation of Eukaryotic Gene Expression

24. Transcription Results in an Unprocessed Message 5 ´ Cap added immediately to 5 ´ sequence, in this case ACATTTG Poly(A) tail added when sequence (5  AATAAA 3  ) is transcribed Heterogeneous nuclear RNA (hnRNA) also known as (aka) pre-mRNA or the primary transcript

25. Translation of mRNA yields Protein

29. A Typical Gene Transcribed by RNAP II

30. Anatomy of a Gene

31. Transcription Initiation Promoters

34. The Promoter Binds General Transcription Factors & RNAP II

35. In vitro Mutagenesis Shows Critical Sequences for Transcription Initiation

38. Enhancer Sequences (Response Elements)

39. Upstream Elements Vary with Gene Function

40. Modular Nature of Upstream Region for Tissue-Specific Gene Expression Note that many different transcription factors can bind to one gene It is the set of proteins bound to a gene that determine the level and location of gene expression

41. Bending DNA Stimulates Transcription

44. General (Basal) TFs

46. Stages of Initiation

48. Assembling the Basal Complex

49. Initiation

50. TAFs Interact with TFIID (& TBP)

51. TBP-DNA TFIIB-TBP-DNA

53. Mediator Complexes Are Composed of Many Coactivators

54. Coactivators Interact with TFs, But Do Not Bind to DNA

57. Multiple Pathways Affect Transcriptional Activation

59. Promoter Escape Requires CTD Phosphorylation by TFIIH

60. Other Proteins Associated with Promoter Escape & Elongation

61. Elongation Phosphorylation of Ser 2 recruits splicing factors Phosphorylation of Ser 5 recruits capping factors Other factors include TFIIS, P-TEFb, TAT-SF1

68. Elongation

69. Transcription Visualized Prokaryotic Eukaryotic

76. Splicing Visualized Transcription initiated here Introns loop out as they are excised

77. Splicing Mechanisms Introns Are Classified by Their Splicing Mechanism

78. Splicing involves two transesterifications

79. Trans-Splicing joins exons from two different RNAs

80. Self-excising group I & 2 introns 2 nucleophilic transesterification reactions 3  -OH guanosine on right side of intron is transferred to nucleotide at 5  end of intron Guanosine acts as cofactor "New" 3  -OH on left side of intron and phosphate group on 3  end of right side of intron interact leaving phosphate for ligation of exons 1 and 2

82. hnRNPs Involved in Splicing Reactions

83. Nuclear Splicing 1. U1 binds to exon 1-intron (5  splice site) binding site 2. U2, U4, U5 & U6 bind, splicing begins (2 trans-esterification reactions) 3. 2  -OH from branchpoint (internal adenine residue) of intron attacks 5  splice site & cuts polymer 4. Free OH created at the end of exon 1 attacks the intron-exon 2 junction 5. Introns excised 6. Exons ligated

84. Assembling the Splicesome

86. Putting It All Together

87. Alternative Splicing Regulates Gene Expression

88. Alternative splicing

89. Alternative splicing results in families of proteins (splicing isoforms)

90. Types of Alternative Splicing (a) Alternative 5 ´ splice site (b) Alternative 3 ´ splice site (c) Skipping the variable alternative splice exon (d) Mutual exclusion of exons (e) Gender-specific splicing Alternative Poly(A) site Found in prostate cancer

91. Preprotachykinin (PPT) Gene P P

92. Splicing is regulated

93. Combinatorial Control Sex lethal binding results in stop codon being spliced out Functional transformer binding causes doublesex to be spliced in a female-specific fashion Stop codon remains Transformer not functional Male-specific doublesex

94. Exon shuffling

95. RNA Editing Changes the sequence of the RNA after transcription, but before translation

97. gRNA Directs T. brucei RNA Editing

99. Apo-B Gene Is Modified by RNA Editing

101. ADAR1 Mechanism

103. RNAi/miRNA Mechanism Kosik Nature Reviews Neuroscience 7 , 911 – 920 (December 2006) | doi:10.1038/nrn2037

104. C. elegans makes lin-4 antisense to regulate lin-14 expression Now consider lin-4 antisense an miRNA

106. Nuclear transport

109. Altering mRNA Stability Allows for Translational Control

110. Prokaryotic Regulation Operons

112. Activation of gene expression Recruitment of polymerase Allosteric activation

113. Cooperative binding and DNA bending activate gene expession

114. Negative Control Gene is expressed unless it is turned off by some regulatory molecule

115. Positive Control Gene only expressed if a regulatory molecule stimulates RNA synthesis

116. The lac Operon Prokaryotic genes do not have introns and exons  make polycistronic mRNA - continuous transcripts that are composed of many genes.

117. Expression of lac genes Control region

118. Negative Control

119. Repressor binds to operator but activator interacts with CTD tail

120. Inducer Changes Repressor Conformation

121. Operators

122. Lac Operon Has 3 Operators All 3 operators must be bound for maximal repression . Repressor binding to 2 operators causes a DNA conformational change  DNA bends away from the repressor forming a repression loop, preventing RNA polymerase access to the promoter

123. Operator Mutations Are Constitutive

124. lacI Mutations Are Constitutive

125. Repressor Mutants Are Super-repressed

126. Catabolite Repression

128. trp operon is repressible

129. Transcription Occurs in the Absence of Tryptophan

130. Repressor Binds in the Presence of Tryptophan

133. Attenuation Is Dependent Upon Leader Sequence

134. trp Leader Sequence

136. Tryptophan Present

138. Low Tryptophan

139. Translation

142. tRNA exhibits atypical basepairing

143. tRNAs exhibit secondary and tertiary structure that results in the formation of loops and stems

146. Synthetase Structure

147. Amino Acids

150. Recognition of correct tRNA

151. Biological polymerization of AA into polypeptide chains

154. Prokaryotic vs. Eukaryotic Ribosome

155. Electron Micrographs of the Ribosome

156. Ribosome Active Sites

157. Mechanisms and Process

158. Transcription and translation are coupled in prokaryotes Overview Polyribosomes

160. Ribosome Has 2 Sites for Binding Charged tRNA

165. fMet Removed During Synthesis

166. Initiation

171. Antibiotics Inhibit Translation

177. eIF4E Binding Proteins Compete for Binding with eIF4G eIF4E, A and G = eIF4F complex Sonenberg & Gingras, 1998

178. Control at initiation by numerous signal transduction pathways Many signal transduction pathways converge on eIF4E and phosphorylate it as well as other IF factors & ribosomal proteins Sonenberg & Gingras, 1998 eIF, Devers, 1999

184. Overview EF-Tu carries aa-tRNA to A site

185. Peptide Bond Formation

186. Translocation

187. Translocation

193. A Translation Movie

194. Translation Animation Web Addresses http://www.geocities.com/CapeCanaveral/Lab/5451/transgif.htm http://tidepool.st.usm.edu/crswr/protsynthmov.html http://www.bio.cmu.edu/Courses/BiochemMols/ribosome/70S.htm http://www.ncc.gmu.edu/dna/ANIMPROT.htm