Mary K. Campbell Shawn O. Farrell �������������������������� Chapter Eleven Chapter Eleven Transcription of the Genetic Code: The Biosynthesis of RNA Paul D. Adams • University of Arkansas 1 Transcription 2
3 Transcription in Prokaryotes • E. coli RNA Polymerase: • molecular weight about 500,000 • four different types of subunits: α , β , β ’, and σ • the core enzyme core enzyme is α 2 ββ ’ • the holoenzyme holoenzyme is α 2 ββ ’ σ • the role of the σ subunit is recognition of the promoter promoter locus locus ; the σ subunit is released after transcription begins • of the two DNA strands, the one that serves as the template for RNA synthesis is called the template strand or antisense antisense strand strand ; the other is called the coding (or nontemplate ) strand or sense strand sense strand • the holoenzyme binds to and transcribes only the template strand 4
The Basics of Transcription 5 Coding strand 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 first nucleotide of gene to be transcribed is • Promoters -DNA sequence that provide direction for RNA polymerase 7 Promoter Sequence � 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? 8
9 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 10
Initiation and Elongation in Transcription 11 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 ribonucleotides • Topoisomerases relax supercoils in front of and behind transcription bubble 12
Chain Elongation (Cont’d) 13 Chain Termination • Two types of termination mechanisms: • intrinsic termination- controlled by specific sequences, termination sites • Termination sites characterized by two inverted repeats 14
Chain Termination (Cont’d) • Other type of termination involves rho ( ρ ) protein • Rho-dependent termination sequences cause hairpin loop to form 15 Transcription Regulation in Prokaryotes • In prokaryotes, transcription regulated by: • alternative σ factors • enhancers • operons • transcription attenuation • transcription attenuation • Alternative σ σ σ σ factors • Viruses and bacteria exert control over which genes are expressed by producing different σ -subunits that direct the RNA polymerase to different genes. 16
Control by Different σ Subunits 17 Operon • 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 • operons are usually not transcribed all the time � β β β -Galactosidase β β β β β 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 18
How Does Repression Work • Repressor protein made by lacI gene forms tetramer when it is translated • Repressor protein then binds to operator binds to operator portion of operon • Operator and promoter together are the control sites 19 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 20
Binding Sites On lac operon (Cont’d) 21 Catabolite Repression • CAP forms complex with cAMP • Complex binds at CAP site • RNA polymerase binds at available binds at available binding site, and transcription occurs 22
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 enhancers • Bound by proteins called transcription factors 23 Elements of a Bacterial Promoter 24
Basic Control Mechanisms in Gene Control • Control may be inducible or repressive , and these may be negatively or positively controlled 25 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 26
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 27 Attenuation in the trp operon • Pause structure forms when ribosome passes over Trp codons when Trp levels are high high • Ribosome stalls at the Trp codon when trp levels are low and antiterminator loop forms 28
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 • 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 29 RNA Polymerase II • Most studied on the polymerases • Consists of 12 subunits • RPB- RNA Polymerase B 30
How does Pol II Recognize the Correct DNA? • Four elements of the Pol II promoter allow for this phenomenon 31 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 32
General Transcription Initiation Factors 33 Transcription Order of Events • Less is known about eukaryotes than prokaryotes • The phosphorylated Pol II synthesizes RNA and leaves the RNA and leaves the promoter region behind • GTFs are left at the promoter or dissociate from Pol II 34
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) negative transcription elongation factor (N-TEF) • Termination • begins by stopping RNA Pol; the eukaryotic consensus sequence for termination is AAUAAA 35 Gene Regulation • Enhancers and silencers- regulatory sequences that augment or diminish transcription, respectively • DNA looping brings enhancers into contact with transcription factors and polymerase 36
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 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 CRE – cAMP-response element GTFs) activates CREB – cAMP-response element binding protein transcription CBP – CREB-binding protein PKA – cAMP dependent protein kinase (protein kinase A) 38
Response Elements 39 Non-Coding RNAs • As much as 98% of transcriptional output from human genomes may be comprised of non-coding RNAs (ncRNA) • Linked to: regular transcription, gene silencing, replication, processing of RNA, RNA modification, translation, protein stabilization, protein translocation translation, protein stabilization, protein translocation • Two main types: Micro RNA (miRNA), and Small Interfering RNA (siRNA) 40
SiRNAs are formed in away similar miRNA 41 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 • DNA-Binding domains have domains that are either: • Helix-Turn-Helix (HTH) • Zinc fingers • Basic-region leucine zipper 42
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