TRANSCRIPTION (DNA → mRNA)

Fig. 17-7a-2 Promoter Transcription unit DNA Start point RNA polymerase Initiation RNA transcript 5 5 Unwound DNA Template strand of DNA 3 STAGES OF TRANSCRIPTION

Link to Transcription factors animation

Fig. 17-7a-3 Promoter Transcription unit DNA Start point RNA polymerase Initiation RNA transcript 5 5 Unwound DNA Template strand of DNA 2 Elongation Rewound DNA RNA transcript 3 STAGES OF TRANSCRIPTION

Fig. 17-7a-4 Promoter Transcription unit DNA Start point RNA polymerase Initiation RNA transcript 5 5 Unwound DNA Template strand of DNA 2 Elongation Rewound DNA RNA transcript 3 Termination Completed RNA transcript 3 STAGES OF TRANSCRIPTION

TRANSCRIPTION INITIATION  START OF TRANSCRIPTION REQUIRES FORMATION OF TRANSCRIPTION INITIATION COMPLEX COMPOSED OF DNA AND PROTEIN COMPLEX THAT INCLUDES RNA POLYMERASE (in eukaryotes, transcription factor proteins must also be present) FORMATION OF TRANSCRIPTION INITIATION COMPLEX COMPOSED OF DNA AND PROTEIN COMPLEX THAT INCLUDES RNA POLYMERASE (in eukaryotes, transcription factor proteins must also be present)

Important DNA regions for gene expression and formation of transcription initiation complex  Gene – segment of DNA that codes for protein  Promoter – 25 to 200 bp segment upstream of gene to which RNA polymerase binds; overlaps start point of gene; In Eukaryotes, promoter is binding site for transcription factor proteins before RNA polymerase can bind.  Enhancer regions – segments of DNA far upstream or downstream of gene (but which may be physically close due to DNA being wrapped around histones) which are binding sites activator proteins; activator proteins are required to initiate transcription.

Fig A eukaryotic promoter includes a TATA box Promoter TATA box Start point Template DNA strand Transcription factors Several transcription factors must bind to the DNA before RNA polymerase II can do so Additional transcription factors bind to the DNA along with RNA polymerase II, forming the transcription initiation complex. RNA polymerase II Transcription factors RNA transcript Transcription initiation complex

Link to Transcription factors animation Link to Transcription factors animation

Fig. 17-7b Elongation RNA polymerase Nontemplate strand of DNA RNA nucleotides 3 end Direction of transcription (“downstream”) Template strand of DNA Newly made RNA 3 5 5

Termination of Transcription  Bacteria – terminator sequence triggers release of mRNA  Eukaryotes- i) polyadenylation (AAUAAA) signal in pre- mRNA i) polyadenylation (AAUAAA) signal in pre- mRNA ii) bp downstream of AAUAAA sequence, pre-mRNA transcript released ii) bp downstream of AAUAAA sequence, pre-mRNA transcript released

Review Questions: Transcription  1) What is transcription?  ANS: Process of making mRNA from DNA  2) Name 3 phases of Transcription  ANS: Initiation, Elongation, Termination  3) What is the promoter region?  ANS: 25 to 200 bp segment upstream of gene to which RNA polymerase binds; overlaps start point of gene

Transcription Review Continued  What is a transcription factor?  ANS: A protein found primarily in eukaryotes that binds to the promoter on the DNA and binds to RNA polymerase. In Eukaryotes, RNA polymerase will not bind to the promoter by itself without transcription factors.  What is the enhancer region?  ANS: segments of DNA far upstream or downstream of gene which are binding sites activator proteins; activator proteins are required to initiate transcription.

Put these events in the correct order.  A) Activator proteins bind to the enhancer region.  B) Activator protein/enhancer complex binds to RNA/transcription factor initiation complex; this event initiates transcribing of the gene.  C) Transcription factors bind to the promoter.  D) RNA polymerase binds to the transcription factors forming the transcription initiation complex.  ANS: C, D, A, B

Transcription Termination Review  Which termination mechanism is used by prokaryotes and which is used by eukaryotes?  A) i) polyadenylation (AAUAAA) signal in pre-mRNA ii) bp downstream of AAUAAA sequence, pre- mRNA transcript released ii) bp downstream of AAUAAA sequence, pre- mRNA transcript released  B) terminator sequence triggers release of mRNA  ANS: PROKARYOTES = B; EUKARYOTES = A

mRNA processing  Processing only occurs in Eurkaryotes within nucleus; 3 major parts  1) Intron removal by splicing  2) 5’ Cap -Add modified a Guanosine base to 5’ end of mRNA  3) Poly A tail – 50 to 250 Adenine nucleotides added to 3’ end

Figure 17.9 RNA processing: RNA splicing

Figure Correspondence between exons and protein domains

Evolutionary Advantage to splicing?  Hypothesis: Different exons code for specific domains.  Example: Domain 1 contains active site  Domain 2 contains allosteric site regulating enzyme activity in response to specific chemical signal  By replacing domain 2 with a different domain that responds to different signal produce “new” enzyme which catalyzes same reaction but responds to different allosteric signals

Diagram for Changing Domains

Fig RNA transcript (pre-mRNA) Exon 1Exon 2Intron Protein snRNA snRNPs Other proteins 5

Fig RNA transcript (pre-mRNA) Exon 1Exon 2Intron Protein snRNA snRNPs Other proteins 5 5 Spliceosome

Fig RNA transcript (pre-mRNA) Exon 1Exon 2Intron Protein snRNA snRNPs Other proteins 5 5 Spliceosome components Cut-out intron mRNA Exon 1 Exon 2 5

Link to RNA splicing animation

Alternation of mRNA ends; Addition of 5’ Cap and Poly A tail Facilitates export of processed mRNA out of nucleus Helps protect mRNA from enzymatic degradation in cytosol “Attach here” signal for ribosomes

RNA Processing Review Questions  1) Why doesn’t processing take place in prokaryotes?  ANS: Prokaryotes lack the nuclear membrane necessary to physically separate transcription from translation  2) List the 3 major events of RNA processing:  ANS: i) RNA splicing ii) Addition of 5’ CAP iii) Addition of Poly – A tail

RNA Processing Review Continued  Which region the exon or the intron represents the coding region and which region is spliced out?  ANS: Introns are removed, Exons regions are translated.  What is the name of the complex responsible for splicing the mRNA? What is the complex made of ?  ANS: The splicesome; It is composed of RNA and protein. The RNA catalyzes the splicing reactions.

RNA processing continued  What is the hypothesis to explain why the cell “throws away” a large segment of the RNA transcribed from the DNA?  ANS: The leading hypothesis is that splicing makes it easy to develop new proteins needed to survive under new environmental conditions. According this idea, exons represent different functional regions of the protein. Different splicing patterns can combine functional units that comprise a protein in new ways.

RNA Processing Continued  State 3 roles for the 5’ CAP and Poly A tail in the cell.  ANS:  1) Facilitates export of processed mRNA out of nucleus  2) Helps protect mRNA from enzymatic degradation in cytosol  3) “Attach here” signal for ribosomes

Figure Translation: the basic concept