Transcription process

Biochemistry & Molecular chemistry

1. Transcription Process

2. Transcription: Synthesis of mRNA Transcription is the process of synthesis of RNA from DNA During transcription a section of DNA containing the gene unwinds. one strand of DNA bases is used as a template. mRNA is synthesized using complementary base pairing with uracil (U) replacing thymine (T). the newly formed mRNA moves out of the nucleus to ribosomes in the cytoplasm. 2

3. RNA Transmits information from DNA to make proteins. has several types Messenger RNA (mRNA) (5-10%) carries genetic information from DNA to the ribosomes . Transfer RNA ( tRNA ) (10-15%) brings amino acids to the ribosome to make the protein. Ribosomal RNA ( rRNA ) (75%) makes up 2/3 of ribosomes where protein synthesis takes place. In prokaryotic cell: 23S, 16S 5S In eukaryotic cell: 28S, 18S 5.8S, 5S 3

4. tRNA Each tRNA has a triplet called an anticodon that complements a codon on mRNA. bonds to a specific amino acid at the acceptor stem. 4 Anticodon

5. Steps in RNA synthesis A. Initiation: Initiation of transcription involves the binding of RNA polymerase to a region on the DNA that determines the specificity of transcription of that particular genes. This region is known as promoter region . The characteristic nucleotide sequencesof the prokaryotic promoter region that are recognized by RNA polymerase include: ( i ) Pribnow box: consists of 6 nucleotides TATAAT centered at 8-10 nucleotides to upstream of the transcription start site that codes the initiation codon on mRNA 5

6. 6 -35 sequence: A second nucleotide sequence, TTGACA, located at 35 bases upstream of transcription start site is also recognized by RNA polymerase. Properties of RNA polymerase (Prokaryotic cell): It consists of four peptide subunits (2  , 1 β , 1 β / ). It is responsible for the 5 / →3 / RNA polymerase activity. This enzyme lacks specificity. It is core e nzyme . The σ subunit enables the polymerase to recognize promoter region on the DNA. The σ subunit and the core enzyme make up the holo enzyme.

7. B. Elongation: Once the promoter region has been recognized by the holoenzyme , its binding with the DNA template results in a local unwinding of the DNA helix. After binding RNA polymerase begins to synthesize a transcript of the DNA sequence and σ subunit is released. RNA polymerase does not require a primer and has no endo - & exo -nuclease activity. For this it has no ability to repair mistakes in the RNA. RNA polymerase utilizes ribonucelside triphosphate and releases pyrophosphate each time a nucleotide is added to the growing chain. 7

8. C. Termination: The process of elongation of the RNA chain continues until a termination signal is reached. There are two types of termination signals. ρ -Independent termination: It requires that the newly synthesized RNA has two important structural features. First: the RNA transcipt must be able to form a stable hairpin that slows down the progress of RNA polymerase and causes it to pause temporarily. Near the bases of the stem of hairpin, a sequence occurs that is rich in C and G. Second: following the hairpin turn, the RNA transcript contains a strings of Us. The bonding of U to the DNA’ A is very week that facilitates the separation of RNA. 8

9. ρ -Dependent termination: It requires the participation of an additional factor which has an RNA-dependent ATPase activity. Inhibition of Transcription/Action of Antibiotics: - Rifampicin inhibits transcription by binding to the of β -subunit of RNA polymerase (prokaryotic). - Dactinomycin binds to DNA template and interfere with the movement of RNA polymerase along the DNA (eukaryotic) 9

10. Steps in RNA synthesis (Eukaryotic Cell) RNA polymerase I: synthesizes the precursor of large ribosomal RNAs (28S, 18S, 5.8S). RNA polymerase II: synthesizes precursor of mRNA. Promoters: - TATA box (ATATAA) at 25 nucleotides of upstream of initiation base pair. - CAAT box (GGCCAATC) at around 70-80 nucleotides of upstream of initiation base pair. Enhancer: It is DNA sequence that increase the rate of transcription by RNA polymerase II. They can be located at upstream or downstream of the gene and can be close to or thousands of base pairs away from the promoter. It binds with proteins that interact with transcription factors bound to promoters thereby affecting transcription. 11

11. Post Transcriptional Modification Ribosomal RNA: After synthesis (45S) it split into small fragments (23S, 16S, 5S for prokaryotes and 28S, 18S, 5.8S for eukaryotes) by RNases . In both prokaryotic and eukaryotic cell, tRNAs are made from longer precursors. 5’ capping in eukaryotic cell: It is a 7-methylguanosine attached backward through a triphosphate to the 5’ terminal end of the RNA. It helps to stabilize RNA and to translate to protein. Addition of poly A tail in eukaryotic cell: 40-200 adenine nucleotides attached to the 3’-end of the RNA. It helps to stabilise mRNA and to transfer through nuclear membrane. Splicing in eukaryotic cell : Removal of introns 12

12. Genetic Code The genetic code is a sequence of amino acids in a mRNA that determine the amino acid order for the protein. consists of sets of three bases (triplet) along the mRNA called codons . has a different codon for all 20 amino acids needed to build a protein. contains certain codons that signal the “start” and “end” of a polypeptide chain. 16

13. The Genetic Code: mRNA Codons 17

14. Codons and Amino Acids Determine the amino acids from the following codons in a section of mRNA. — CCU — AGC — GGA — CUU — According to the genetic code, the amino acids for these codons are CCU = proline AGC = serine GGA = glycine CUU = leucine This mRNA section codes for an amino acid sequence of — CCU — AGC — GGA — CUU — — Pro — Ser — Gly — Leu — 18