Lecture 9 Gene Expression in Prokaryotes and Eukaryotes PDF

Recombina nt DNA202 Gene Expression: Transcriptio n RDNA202 Cassie [email protected] GENE Expression in Prokaryotes and Eukaryotes Lecture 9: GENE Expression – Transcription Lecture 10: GENE Expression – Translation Lecture 11: DNA Mutation  Learning Outcomes T...

Recombina nt DNA202 Gene Expression: Transcriptio n RDNA202 Cassie [email protected] GENE Expression in Prokaryotes and Eukaryotes Lecture 9: GENE Expression – Transcription Lecture 10: GENE Expression – Translation Lecture 11: DNA Mutation  Learning Outcomes Transcription Poly-A-Tail In prokaryotes Intron In Eukaryotes Exon Promotor Steps in Transcription RNA polymerase Initiation Holoenzyme Elongation Splicing Termination Spliceosomes Pre-mRNA modification  The Central Dogma The process by which genetic information in the cell flows from DNA to mRNA to Proteins Process occurs in two stages: 1. Transcription 2. Translation  Basic Principles of Transcription and Translation Instructions for protein synthesis comes from genes RNA serves as the bridge between DNA and Protein synthesis RNA contains Uracil not Thymine DNA has A, C, G, or T RNA has A, C, G, or U  Transcription Synthesis of mRNA Uses genetic information encoded in DNA DNA strand serves as the template strand for the synthesis of RNA nucleotides (mRNA) NOTE: mRNA molecule will be complementary to the DNA template Identical to the non-template DNA (with the exception of uracil)  Which Strand of the DNA is copied? DNA is double stranded… BUT In any gene – only ONE strand is copied into RNA  Stages of Transcription 3 Stages of Transcription: 1. Initiation 2. Elongation 3. Termination  Transcription in Prokaryotes: Initiation Promotor – DNA sequence where RNA Polymerase attaches & initiates transcription 2 Sequences Start point – the nucleotides where RNA polymerase begins synthesising mRNA RNA Polymerase Separates 2 DNA strands Joins together complementary RNA nucleotides to DNA template strand  Transcription in Prokaryotes: Initiation Prokaryotic RNA Polymerases The same RNA polymerase is used to transcribe all the genes in prokaryotes Comprises five polypeptide subunits 1. β 2. β’ 3. α 4. α 5.  The enzyme that comprises all five subunits - holoenzyme  Transcription in Prokaryotes: Initiation Each Subunit has a unique function The β-subunit binds to the ribonucleoside triphosphate that will become part of the new mRNA The β’-subunit binds the DNA template strand The two α-subunits are required to assemble the polymerase on the DNA The -subunit is involved in transcription initiation Causes the polymerase to synthesize mRNA from an appropriate initiation site  Transcription in Prokaryotes: Initiation At the -10 and -35 regions upstream of the start point – there are 2 promoter consensus sequences The  -subunit attaches to the sequences for RNA synthesis to begin RNA polymerase unwinds the DNA strand and initiates transcription Once transcription has been initiated,  -subunit dissociates from the polymerase  Transcription in Prokaryotes: Elongation During elongation the  -subunit is released from the polymerase Causes the enzyme to proceed with synthesizing mRNA in 5’-3’ direction Adds RNA nucleotides to the 3’ end of the growing strand  Transcription in Prokaryotes: Termination Prokaryotic polymerase needs to be instructed/signalled to dissociate from the DNA template Set free the newly made mRNA The transcribed (produced) RNA sequence serves as the termination signal Causes RNA polymerase to detach from DNA & release transcript The RNA transcript does not require further modification or processing before translation  Transcription in Eukaryotes: Initiation Prokaryotic polymerase – can bind to DNA template on its own Eukaryotic polymerase cannot bind to DNA template on its own Eukaryotes require transcription factors These first bind to the promotor region Help recruit the appropriate polymerase Both have similar sequences at the -10 region TATAAA in eukaryotes TATAAAT in prokaryotes  Transcription in Eukaryotes: Initiation In the promotor region there is a TATA box & Transcription start point Essential in forming initiation transcription complex The nucleotide position where RNA polymerase begins RNA synthesis  Transcription in Eukaryotes: Initiation Transcription factors (purple) bind to DNA RNA polymerase II can only bind to promotor after transcription factors are attached RNA polymerase II is bound to transcription factors on DNA RNA polymerase separates DNA strands & begins RNA synthesis  Transcription in Eukaryotes: Elongation As RNA polymerase moves along DNA – untwists double helix RNA polymerase adds RNA nucleotide to 3’ end of growing RNA strand Newly synthesized RNA molecule peels away from DNA template as transcription progresses  Transcription in Eukaryotes: Termination RNA polymerase II transcribes a sequence on DNA – Polyadenylation signal sequence (AAUAAA) Once these 6 nucleotides appear – bound to nucleus by certain proteins These proteins release the pre-mRNA The pre-mRNA then undergoes processing  Pre-mRNA Processing in Eukaryotes Mechanism of transcription is similar in bacteria and eukaryotes But… The flow of genetic information differs within the cells  Pre-mRNA Processing in Eukaryotes In Bacteria: In Eukaryotes: Bacteria do not have nuclei Do have nuclei No nuclear membrane to Nuclear envelope separates separate DNA & mRNA from ribosomes transcription from translation Translation of mRNA occurs Transcription – in nucleus without additional processing mRNA must be transported to Both occur in cytoplasm cytoplasm for translation  Pre-mRNA Processing in Eukaryotes Before RNA transcript (pre-mRNA) can leave nucleus Must undergo processing to produce final mRNA Transcription produces pre-mRNA Further pre-mRNA processing results in finished mRNA Transported to cytoplasm for translation  Pre-mRNA Processing in Eukaryotes Three steps involved in pre-mRNA processing 1. Addition of stabilizing and signalling factors and 5’ and 3’ ends of molecule 5’ capping Poly-A-Tail 2. Removal of introns – mRNA splicing 3. mRNA transcripts can be edited after it is transcribed – in rare cases  Pre-mRNA Processing in Eukaryotes 1. Addition of stabilizing and signalling factors at the 5’ and 3’ ends 5’ capping A 7-methylguanosine cap (form of guanine nucleotide) – added to 5’ end Protects mRNA from degradation During protein synthesis – factors recognise this cap to help initiate translation  Pre-mRNA Processing in Eukaryotes 1. Addition of stabilizing and signalling factors at the 5’ and 3’ ends 3’ Poly-A-Tail Pre-mRNA is cut and released soon after the Poly-A-Tail appears At 3’ end – enzyme (Poly A Polymerase) adds more adenine nucleotides (50-250) This protects pre-mRNA from degradation Also serves as binding site for protein exporting processed mRNA into cytoplasm  Pre-mRNA Processing in Eukaryotes 2. Pre-mRNA splicing: The removal of introns Process where large portions of RNA transcript are removed & remaining portions re-connected Intron: Non-coding sequence within primary transcript Removed from transcript during RNA processing Exon: Sequence within primary transcript Remains in RNA after RNA processing Pre-mRNA splicing is conducted by spliceosomes  Pre-mRNA Processing in Eukaryotes During pre-mRNA processing Both ends of primary transcripts are altered/modified Includes: Cutting out of introns Joining together Exons Alteration of 5’ and 3’ ends This stage of RNA processing is called RNA splicing  Pre-mRNA Processing in Eukaryotes How is Pre-mRNA splicing carried out? 1. Spliceosomes recognise 5’ and 3’ end of intron 2. Small RNAs in spliceosome pairs with the nucleotide of the intron 3. Small RNAs catalyze cutting of the pre-mRNA & joining of the exons  Next Lecture: GENE EXPRESSION – TRANSLATION