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RNA Synthesis (Transcription) Dr. Shumaila Asim Lecture # 4 1 RNA Synthesis (Transcription) Introduction First step in gene expression RNA is involved in the transfer of genetic information stored in DNA to proteins Process of DNA directed RNA synthesis (copying process) (genes in DNA make RNA copies) Synthesis of an RNA strand with a base sequence complementary to one strand of DNA (template) and identical in sequence to the other strand 2 RNA Synthesis (Transcription) Selective process; one particular gene or groups of genes are transcribed Specific sequences indicate the beginning and end of DNA segment to be transcribed Some proteins are also involved in this “selectivity of the process” 3 RNA Synthesis (Transcription) Occurs in 5 − 3 direction Catalyzed by RNA polymerase I, II & III (DNA dependent RNA polymerases (RNAPs) Each RNAP consists of large subunits and many small subunits 4 two Comparison of Replication & Transcription S.No 1 2 3 4 5 5 Replication Transcription DNA template required DNA template required Whole genome is copied Initiation & termination signals not required RNA primer is required Selective parts of DNA are copied Initiation & termination signals required Primer is not required Synthesized DNA strand remains attached to template strand RNA does not remain attached to template DNA strand Comparison of Replication & Transcription S.No 6 Replication Transcription 6 Deoxy-ribonucleotides are Ribonucleotides are required required 7 DNA polymerases 8 Very much accurate (exact) Less accurate 9 No changes required after Post-transcriptional replication changes required RNA polymerases Numbering -5 -4 -3 -2 -1 +1 +2 +3 +4 +5 +6 DNA RNA A –T – A – C – G – T – T – G – A – C – G ----A – A – C – U – G – C ----- Position +1 of a gene is the base that is complementary to the first base of the 5 end of the RNA transcript of that gene (initiation point) Sequences preceding first base are numbered negatively and are said to be upstream of the initiation point Sequences following the first base are numbered positively – 7 downstream Specific Sequences a. Promoters – Specific areas or sequences of DNA for initiation (different for different RNAPs) ,these are 8 Pribnow box (TATA box) (Prokaryotes) Goldberg-Hogness box or TATA box or consensus sequence (Eukaryotes) Specific Sequences b. Terminators – Sequences for termination c. Enhansers & silencers – Sequences for increasing or decreasing rate of transcription d. Transcription Unit – Region between promoter and terminator (includes sequences for initiation, elongation & termination) 9 Requirements Template = DNA single strand Substrates = Ribonucleotides (ATP, GTP, UTP, CTP) Initiation factors Transcription factors [Tf – II or GTF (General Transcription factors)] (Tf-II include Tf-IIA, Tf-IIB, Tf-IIC, Tf-IID, Tf-IIE, Tf-IIF, Tf-IIH) Tf-IID contains TATA binding protein (TBP) and 14 TAFs (Transcription activating factors) Activators, Co-activators and components of PIC (pre-initiation complex) 10 Requirements Enzymes = DNA-dependent RNA polymerases [RNA polymerase (RNAP) I, II, III] RNAP–I synthesizes rRNA RNAP–II synthesizes mRNA RNAP–III synthesizes tRNA, 5S rRNA Each RNAP holoenzyme consists of Core-enzyme + one or more sigma ()factors Sigma () factors 11 the core enzyme to recognize promoter region assist Requirements RNAP is zinc containing metallo-enzyme, requires Mg++ ions for its activity Core enzyme consists of two identical -subunits, , and subunits -subunit is thought to be the subunit (the growing chain of RNA remains 12 attached to this subunit) one each catalytic Generalized Functions of RNAPs Unwinding the DNA in front, Synthesis of RNA, Re-winding it behind, Dissociating the growing RNA chain from the template and Perform proofreading. 13 RNA Synthesis (Transcription) 14 A cyclic process, involving RNA chain initiation, elongation & termination RNA polymerase attaches to and initiates transcription at a distinct site, the promoter (a special DNA sequence) GTFs (Tf-II) facilitate promoter-specific binding of RNAPs to form pre-initiation complex (PIC) Other proteins (co-activator proteins) regulate the rate of transcription initiation RNA Synthesis (Transcription) 15 RNA Synthesis (Transcription) 16