Transcription and Translation PDF

Transcription and Translation Transcription : o Occurs in the cytoplasm of prokaryotes o Occurs in the nucleus of eukaryotes o DNA is the template to form mRNA o Occurs in 3 steps ▪ Initiation RNA polymera...

Transcription and Translation Transcription : o Occurs in the cytoplasm of prokaryotes o Occurs in the nucleus of eukaryotes o DNA is the template to form mRNA o Occurs in 3 steps ▪ Initiation RNA polymerase complex (holoenzyme) binds to the promotor site The sigma subunit helps DNA unwind which allows RNA polymerase to begin RNA synthesis ▪ Elongation RNA polymerase moves along the DNA template strand in the 3’ to 5’ direction, synthesizing the RNA strand in the 5’ to 3’ direction RNA polymerase catalyzes the formation of phosphodiester bonds between adjacent ribonucleotides, elongating the RNA strand. The enzyme ensures that the correct ribonucleotide is added by complementary base pairing. o The β subunit contains the polymerase activity that catalyzes the synthesis of RNA. It is responsible for the addition of ribonucleotides to the growing RNA chain, ensuring that the RNA strand is synthesized in the 5’ to 3’ direction RNA polymerase has a proofreading function to ensure the accuracy of RNA synthesis. If an incorrect nucleotide is incorporated, the enzyme can backtrack, remove the incorrect nucleotide, and resume synthesis. Elongation continues until RNA polymerase encounters a termination signal, which triggers the end of transcription and the release of the newly synthesized RNA molecule. ▪ Termination After the synthesis of mRNA o Cleavage and Polyadenylation: RNA Polymerase II transcribes past the end of the gene and the RNA transcript is cleaved at a specific site. A polyadenylation signal sequence (AAUAAA) in the RNA transcript signals for the addition of a poly(A) tail. Post-Transcription Modifications: o 1. Capping ▪ 5’ Cap Addition: In eukaryotes, a modified guanine nucleotide (7- methylguanosine) is added to the 5’ end of the nascent RNA transcript. This cap protects the RNA from degradation and is involved in the initiation of translation. o 2. Splicing ▪ Removal of Introns: Eukaryotic genes often contain non-coding regions called introns. These introns are removed from the pre-mRNA by a process called splicing. ▪ Exon Joining: The remaining coding regions, called exons, are joined together to form a continuous coding sequence. This process is carried out by a complex called the spliceosome. o 3. Polyadenylation ▪ Poly(A) Tail Addition: A string of adenine nucleotides (poly(A) tail) is added to the 3’ end of the RNA transcript. This tail enhances the stability of the RNA and aids in its export from the nucleus to the cytoplasm.  o 4. RNA Editing ▪ Base Modification: In some cases, the nucleotide sequence of the RNA is altered through a process called RNA editing. This can involve the insertion, deletion, or substitution of nucleotides, resulting in a different RNA sequence than originally transcribed from the DNA. o 5. RNA Transport ▪ Nuclear Export: The mature RNA is transported from the nucleus to the cytoplasm, where it can be translated into protein. This transport is facilitated by specific nuclear export signals and transport proteins. o 6. RNA Stability and Degradation ▪ Regulation of RNA Lifespan: The stability and degradation of RNA molecules are tightly regulated. Certain sequences and structures within the RNA can influence its stability, determining how long it remains available for translation. o 7. Alternative Splicing ▪ Generation of Multiple Isoforms: Through alternative splicing, a single gene can produce multiple RNA isoforms, leading to the production of different proteins from the same gene. This increases the diversity of the proteome. Prokaryotic RNA Polymerase: o Core enzyme consists of five subunits: ▪ α (alpha): Two subunits, involved in enzyme assembly and binding to the promoter. ▪ β (beta): Forms part of the catalytic center. ▪ β’ (beta prime): Also part of the catalytic center. ▪ ω (omega): Stabilizes the enzyme. o Holoenzyme – core enzyme plus the sigma factor which is crucial for initiating transcription by recognising the promoter Eukaryotic RNA Polymerase o RNA Polymerase I: Synthesizes rRNA. o RNA Polymerase II: Synthesizes mRNA and some snRNA. o RNA Polymerase III: Synthesizes tRNA and some other small RNAs. Each of these polymerases is composed of multiple subunits The part of DNA being transcribed is called the transcription unit. It is composed of three key regions: o Promoter ▪ A sequence upstream of the gene that signals the start of transcription. RNA polymerase and transcription factors bind here to initiate transcription. o Coding region ▪ The actual sequence of DNA that is transcribed into RNA. This includes exons (coding sequences) and introns (non-coding sequences) in eukaryotes. o Terminator ▪ A sequence downstream of the gene that signals the end of transcription, causing RNA polymerase to release the newly synthesized RNA molecule. Direction of transcription is downstream Translation: Translation is the process by which the genetic code carried by messenger RNA (mRNA) is decoded to produce a specific protein. This process occurs in the cytoplasm of the cell and involves several key steps and components: Key components: o mRNA (Messenger RNA): Carries the genetic information from DNA to the ribosome, where it serves as a template for protein synthesis. o Ribosomes: Molecular machines composed of ribosomal RNA (rRNA) and proteins. They facilitate the assembly of amino acids into protein chains. o tRNA (Transfer RNA): Adapters that carry specific amino acids to the ribosome. Each tRNA has an anticodon that pairs with a complementary codon on the mRNA. o Amino Acids: The building blocks of proteins. There are 20 different amino acids that are assembled in a specific sequence to form a protein. Steps of Translation: o Initiation: ▪ The small ribosomal subunit binds to the mRNA near the start codon (AUG). ▪ The initiator tRNA, carrying methionine, binds to the start codon.  ▪ The large ribosomal subunit joins the complex, forming the complete ribosome. o Elongation: ▪ The ribosome moves along the mRNA, reading codons and bringing in the corresponding tRNA with the appropriate amino acid. ▪ Each new amino acid is added to the growing polypeptide chain through the formation of peptide bonds. ▪ The ribosome has three sites: the A site, the P site, and the E site. The tRNA moves through these sites during elongation. o Termination: ▪ When the ribosome reaches a stop codon (UAA, UAG, or UGA), no corresponding tRNA can bind. ▪ Release factors bind to the ribosome, causing the release of the newly synthesized polypeptide chain. ▪ The ribosomal subunits dissociate, ready to initiate another round of translation. Post-Translational Modifications o After translation, the newly synthesized protein may undergo several modifications, such as: ▪ folding, ▪ cleavage, ▪ addition of functional groups, to become fully functional. o Translation is a highly regulated process, ensuring that proteins are synthesized accurately and efficiently according to the genetic instructions encoded in the mRNA.