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TRANSCRIPTION 1) No free 3'OH end needed for initiation. 2) No primer needed. 3) 5' 3' synthesis directionality. 4) DNA-dependent RNA polymerase copies from one single-stranded region of DNA (antisense strand). 5) Ribonucleoside triphosphate precursors. 6) DNA-RNA duplex less stable than DNA-DNA duplex. RNA falls away from transcription site. Coding or Sense Strand ________________________________________ 3' 5' Has same sequence as mRNA transcript (except that is possesses T instead of U) DNA Template or Antisense Strand ________________________________________ 3' 5' Directs synthesis of complementary mRNA transcript transcription mRNA 5' ________________________________________ 3' Triphosphate precursors Transcription (ribonucleoside triphosphate precursors; rNTPs ) 2P UTP UMP 2P CTP CMP 2P GTP GMP 2P ATP AMP Differences in Gene Expression Between Prokaryotes and Eukaryotes Prokaryotes Eukaryotes 1.Unwinding by gyrases and helicases, but no single strand binding proteins. 1. Possible unwinding by gyrases and helicases, but no single strand binding proteins. 2. All RNA species are synthesized by a single RNA polymerase. 2. Three different RNA polymerases are responsible for the different classes of RNA molecules. 3. mRNA is translated during transcription (coupled). 3. Translation is independent from transcription. Prokaryotes Eukaryotes 4. First nucleotide is a purine. 4. mRNA is processed before transport to the cytoplasm, where it is translated. Caps and tails are added, and internal portions of transcript are removed (splicing). 5. Genes are contiguous segments of DNA that are colinear with the mRNA that is translated into a protein. 5. Genes are often split. They are not contiguous segments of coding sequences rather, the coding sequences are interrupted by intervening sequences (introns). 6. mRNAs are often polycistronic. 6. mRNAs are often monocistronic. 205 A. Prokaryotic – Ex. lac operon model. I = Repressor gene lac = lactose P = Promoter O = Operator 3’ RNA polymerase (Holoenzyme) = core unit + σ (sigma unit) = 5’ σ termination σ70 = vegetative, for all RNAs σ60 = nitrogen metabolism σ 32 = heat shock proteins Initiation: a) Holoenzyme binds to promoter site, as specified by sigma subunit and unwinds short stretch of DNA. b) Sigma subunit released and transcription begins past promoter site. Termination: a) rho-independent: rich G-C dyad region followed by series of A’s and U’s (6-8) on RNA transcript causes formation of hairpin structure and consequently causes RNA polymerase to be knocked off. 5' b) rho-dependent: rho protein (hexamer) binds with RNA transcript at rut site and causes it to fold around it, and upon transclocation and further wrapping pulls the transcript away from the polymerase. B. EukaryoticDifferent RNA polymerases, with no sigma units: RNA polymerase I - rRNA RNA polymerase II - pre-mRNA RNA polymerase III - tRNA and other small structural RNAs Eukaryotic RNA Polymerase RNA polymerase I (inside nucleolus) - makes structural RNA (i.e., rRNA). RNA polymerase II (in nucleoplasm) - makes heterogenous nuclear RNAs or hnRNAs (i.e., precursor mRNAs or premRNAs). RNA polymerase III (in nucleoplasm) - makes small adaptor RNAs (i.e., small nuclear RNAs and tRNAs). Only one RNA polymerase in prokaryotes. However, its activity is much like that of RNA polymerase II in eukaryotes. Initiation: interact (binding) Trans-acting regulatory proteins 1) Transcription Factors (TFs) function in the positive regulation of transcription but are not part of RNA polymerase (RNA Pol). TFs contain two functional domains: i) DNA binding domain - binds selectively to Promoters and Enhancers. ii) trans- acting domain - involved in protein - protein interactions between TFs and between TFs and RNA Pol in the Promoter. See Fig. 1. 3' Fig. 1 Cis-acting nucleotide sequences of two regulatory elements 1) Promoters - bind TFs and RNA Pol - Promoters are necessary for transcription initiation. 2) Enhancers - only bind TFs. TFs bend or loop the DNA. This brings Distant Enhancers and Promoters into close proximity to form activated transcription complexes increasing the overall transcriptional rate. See Fig. 1. RNA Pol II TF TF 5' Fig. 1 3' RNA Pol II TF TF 5' In the Promoter region, the modular elements, TATA box, CAAT box, and GC box, bind TFs in order to stimulate transcription. See Fig. 2 Fig. 2 Pre-mRNA being synthesized by RNA Pol II Promoter (30 – 120 bp long) – upstream of transcriptional start site GC box CAAT box TATA box 3' GGGCGG GGCCAATC TATAAA -110 bp -70 bp Transcription start site Pre-mRNA 5' gene -30 bp +1 bp Enhancers greatly increase the efficiency of transcription initiation. In contrast to Promoters, Enhancers have the following properties: i) Their positions and orientations need not be fixed, ii) They are at a greater distance from the target gene than are Promoters, and iii) They generically enhance the transcription of surrounding genes. Termination: 1) rRNA (RNA Pol I) - Modified rho-independent. Strange G-C dyad region (many A's and U's intervening), followed by 26 A's, followed by 16 bases of any kind, followed by UCCACCUGGUC, followed by 3 bases, followed by AGGC. 2) pre-mRNA (RNA Pol II) - termination mechanism unknown. Processing begins before transcription ends. Possibly, processing proteins may literally pull pre-mRNA away and force RNA Pol II off of DNA. 3) tRNA (RNA Pol III) - standard rho-independent. Three major aspects of post-transcriptional processing of primary transcripts of pre-mRNAs in eukaryotes. RNA splicing - removal of non-coding intron (intervening) sequences, between coding exon (expressed) sequences. Capping of 5' end (i.e., 7-methylquanosine) and addition of poly A tail to 3' end of pre-mRNA. In eukaryotes, pre-mRNA must associate with proteins to get out of the nucleus and into the cytoplasm. Four major aspects of post-transcriptional processing of tRNAs in eukaryotes. Cut at G on 5' end. Addition of CCA sequence at 3' end (where AA will attach). Base modifications (i.e., thymine, pseudouracil) in looped regions. Introns excised from terminal loop. Aspects of post-transcriptional processing of rRNAs in eukaryotes. Introns removed by autocatalytic activity of EXON regions. 213 Exon 1 Intron A Exon 2 Intron B Exon 3 Pulse-Chase tracking of transcription in Eukaryotes: Transport of RNA to the cytoplasm from the nucleus of eukaryotic cells can be determined through pulse-chase studies using 3H-uridine. If a eukaryotic cell is exposed to radioactive RNA precursor for a few minutes, and the intracellular location of the incorporated radioactivity is determined by autoradiography, almost all of the nascent, labeled RNA is found in the nucleus. If the short exposure “pulse” to the labeled RNA is followed by a period of growth in non-radioactive medium before autoradiography, almost all of the radioactivity is found in the cytoplasm.