RNA Synthesis (Transcription) Dr. Shumaila Asim Lecture # 7

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

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”

RNA Synthesis (Transcription) Occurs in 5 − 3 direction Catalyzed by RNA polymerase I, II & III (DNA dependent RNA polymerases (RNAPs) Each RNAP consists of two large subunits and many small subunits

Numbering -5 -4 -3 -2 -1 +1 +2 +3 +4 +5 +6 DNA A – T – A – C – G – T – T – G – A – C – G ----- RNA 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 – downstream

Specific Sequences Promoters – Specific areas or sequences of DNA for initiation (different for different RNAPs) ,these are Pribnow box (TATA box) (Prokaryotes) Goldberg-Hogness box or TATA box or consensus sequence (Eukaryotes)

Specific Sequences Terminators – Sequences for termination Enhansers & silencers – Sequences for increasing or decreasing rate of transcription Transcription Unit – Region between promoter and terminator (includes sequences for initiation, elongation & termination)

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)

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 assist the core enzyme to recognize promoter region

Requirements RNAP is zinc containing metallo-enzyme, requires Mg++ ions for its activity Core enzyme consists of two identical -subunits, one each ,  and  subunits -subunit is thought to be the catalytic subunit (the growing chain of RNA remains attached to this subunit)

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.

RNA Synthesis (Transcription) 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)

RNA Synthesis (Transcription Steps) Initiation: Formation of transcription bubble Protomer–Polymerase complex (Pre-initiation complex) PIC formation leads to Structural changes (melting or unwinding of ssDNA) producing “bubble” known as “transcription bubble”

RNA Synthesis (Transcription Steps) First nucleotide (always purine ATP or GTP) attaches to enzyme (RNAP) through its 5-end RNAP now catalyzes the formation of a phospho-diester bond between the first nucleotide (free 3-end) and the 5-position of next entering nucleotide

RNA Synthesis (Transcription Steps) Synthesis of RNA continues in 5 − 3 direction in a sequence dictated by DNA template (base pairing rule) anti-parallel to DNA template Pyrophosphate (liberated in each addition) is rapidly degraded to two inorganic phosphates making the RNA synthesis irreversible

RNA Synthesis (Transcription Steps) Elongation: After addition of 10 – 20 ribonucleotides, RNAP undergoes a second structural change Moves away from the “promoter” Progresses along DNA molecule – thereby producing “bubble” (DNA unwinding) along DNA template as RNA synthesis proceeds (elongation)

RNA Synthesis (Transcription Steps) Once the RNA polymerase has synthesized a short stretch of RNA (approximately ten bases), it shifts into the elongation phase. This phase requires further conformational change in RNA polymerase that leads it to grip the template more firmly.

RNA Synthesis (Transcription Steps) Termination: Specific sequences or specific signals are recognized by Rho factor ( factor) of RNAP (in Prokaryotes) Rho factor attaches to DNA; the RNAP cannot move further, Dissociates from DNA and Newly synthesized RNA is released.

RNA Synthesis (Transcription Steps) In eukaryotes, the termination is independent of Rho factor Completed RNA chain and RNAP are released from template RNAP holoenzyme reforms, finds another promoter and synthesis of new RNA starts

Modifications (RNA) in primary transcription Terminal additions, Base modifications, Trimming, Internal segment removal and splicing  lead to    functional RNA molecule

Modifications (RNA) in primary transcription RNA polymerase I synthesizes large ribosomal RNAs in nucleolus RNAP–II synthesizes the precursors of mRNAs RNAP–III synthesizes small RNAs like tRNAs, the small 5S rRNA

Post-transcriptional RNA Processing A. rRNA Processing 45 S Precursor rRNA 5.8 S rRNA 18 S rRNA 28 S rRNA Spacer sequences specific endonucleases 5 S rRNA is directly produced from a 5S separate gene

Post-transcriptional RNA Processing B. mRNA – Needs extensive processing Cap formation (7-methyl-guanylate is added to 5-end) Poly-adenylation at 3-end (Poly A polymerase) Splicing hn RNA m RNA (introns + exons) (exons only) introns

Post-transcriptional RNA Processing C. tRNA a. i. One precursor may contain one or more tRNAs ii. Precursor molecule Sequences removed t RNA(s) b. CCA sequence is added to 3-end c. Some bases may be modified (i.e. some of A, U, G & C bases are methylated, reduced & deaminated) before tRNA can adopt final functional structure

Assignment Draw a well labeled diagram of TRANSCRIPTION BUBBLE …….(10-marks) In your biochemistry practical copy Submit your practical copy in biochemistry laboratory Cover your practical copy with a plain paper and plastic cover Write your NAME and ROLL. No on your copy