BY450 Nature and function of the Gene 2: Gene expression and regulation PDF

BY450 Nature and function of the Gene 2: Gene expression and regulation Dr Nigel Brissett Central dogma of molecular biology DNA – RNA - Protein eukaryotes vs prokaryotes  gene expression: Transcription mRNA processing control Translation...

BY450 Nature and function of the Gene 2: Gene expression and regulation Dr Nigel Brissett Central dogma of molecular biology DNA – RNA - Protein eukaryotes vs prokaryotes  gene expression: Transcription mRNA processing control Translation eukaryotes vs prokaryotes  1. Transcription from DNA to mRNA DNA-dependent RNA polymerases (or RNA polymerases) bacterial RNA polymerase incorporate nucleotides into strand of RNA from a DNA template  Structure of bacterial RNA polymerase consists of six subunits: two  one  one ’ one  one  The  subunit is relatively weakly bound and can dissociate.  subunit green  blue ’ pink  yellow  Prokaryotic transcription 5’ 3’ transcription starts at a transcription halts at a promoter (‘upstream’) terminator (‘downstream’) Prokaryotic promoters - some sequences notice anything?  prokaryotic promoter sequences are highly conserved Promoters have two conserved sequence regions (red): –35 element and -10 element (“Pribnow box”) Polymerase sigma subunit binds specifically to consensus sequences Transcription by E. coli RNA polymerase Polymerase initially binds non-specifically to DNA and migrates along molecule until sigma () interacts with -35 and -10 promotor elements. Polymerase unwinds DNA at initiation site and transcription begins. ctd. Sigma subunit dissociates from the core polymerase which migrates along DNA and elongates the growing RNA chain Continues to a termination point Transcription termination (stem-loop) in prokaryotes Termination is signalled by a G-C rich inverted repeat followed by 7 A residues. A stable ‘stem-loop’ mRNA structure is formed which terminates transcription and mRNA dissociates from DNA template alternatively,…….. Transcription termination (stem-loop) in prokaryotes Termination is signalled by a G-C rich inverted repeat followed by 7 A residues. A stable ‘stem-loop’ mRNA structure is formed which terminates transcription and mRNA dissociates from DNA template Alternatively, transcription of some prokaryotic genes is terminated by a ‘termination protein’ (Rho)  Eukaryotic RNA polymerases (long non-coding) Yeast RNA polymerase II 12-17 subunits, 9 ‘conserved’ b similar to bacterial polymerases RNA Pols I and III: similarities to Pol II but different mechanisms and transcription factors (see below) bacterial polymerase Eukaryotic transcription begins with formation of a RNA polymerase II preinitiation complex multiple transcription factors Protein-DNA interactions RNA polymerase II preinitiation complex and transcription initiation Transcription factor -25 (TFIID) binds to promoter consensus sequences BRE, TATAA etc. TFIID Recognition via TAFs (transcription-associated factors) and TBP (TATA- binding protein)  RNA polymerase II preinitiation complex and transcription initiation Transcription factor (TFIID) -25 binds to promoter consensus sequences BRE, TATAA etc. Recognition via TAFs TFIID (transcription-associated factors) and TBP (TATA- binding protein) RNA polymerase and other transcription RNA polymerase and other transcription factors factors (B,E,F,H) bind to form a pre-initiation (B,E,F,H) bind to form a pre-initiation complex. complex. Mediator Mediator protein binds protein binds to complex. to complex.  RNA polymerase II preinitiation complex and transcription initiation Transcription factor (TFIID) -25 binds to promoter consensus sequences BRE, TATAA etc. Recognition via TAFs TFIID (transcription-associated factors) and TBP (TATA- binding protein) Phosphorylation of C-terminal domain of RNA polymerase and other transcription factors polymerase releases Mediator and other general (B,E,F,H) bind to form a pre-initiation complex. TFs. Polymerase II catalyses RNA synthesis in Mediator protein binds to complex. association with other elongation/processing factors From pre-RNA to mature RNA: mRNA processing Prokaryotes- no mRNA processing (simultaneous transcription/ translation in single compartment) mRNAs are translated by a series of multiple ribosomes called “polysomes” Eukaryotes- mRNA processing before translation post-transcriptional processing of eukaryotic mRNA- 3 stages initial/primary transcript or ‘pre- mRNA’ post-transcriptional processing of eukaryotic mRNA- 3 stages initial/primary transcript or ‘pre- mRNA’ a. 5’ cap post-transcriptional processing of eukaryotic mRNA- 3 stages initial/primary transcript or ‘pre- mRNA’ a. 5’ cap b. 3’ poly A tail post-transcriptional processing of eukaryotic mRNA- 3 stages initial/primary transcript or ‘pre- mRNA’ a. 5’ cap b. 3’ poly A tail c. intron splicing Post-transcriptional mRNA processing I: 5’ capping of mRNA A 5′ cap is formed by the addition of a GTP in reverse orientation* to the 5′ end of the mRNA, forming a 5′-to- 5′ linkage * ‘normal’ nucleotide The added G is phophodiester linkages are 5’- 3’ then methylated at (check what this means if the N-7 position unsure) methyl groups are added to the riboses of the first one or two 5’ Me-G cap: stabilises nucleotides in the mRNA and translation mRNA initiation role Post-transcription mRNA processing II Addition of poly-A tail 1.Polyadenylation signals: upstream and downstream (G-U rich) elements, AAUAAA sequence Post-transcription mRNA processing II Addition of poly-A tail 1.Polyadenylation signals: upstream and downstream (G-U rich) elements, AAUAAA sequence 2.Endonuclease cleaves pre- mRNA 10 to 30 nucleotides downstream of the AAUAAA, usually at a CA sequence  Post-transcription mRNA processing II Addition of poly-A tail 1.Polyadenylation signals: upstream and downstream (G-U rich) elements, AAUAAA sequence 2.Endonuclease cleaves pre- mRNA 10 to 30 nucleotides downstream of the AAUAAA, usually at a CA sequence 3. Poly-A polymerase adds video animation Poly A tail stabilises mRNA + roles in a poly-A tail of TheCell7e translation regulation about 200 As to animation 0802 the 3′ end Post-transcriptional mRNA processing III Intron removal small nuclear ribonuclear particles (snRNPs) made introns removed by a up of small nuclear RNAs spliceosome complex (snRNAs) + 6-10 protein molecules (U1, U2, U4, U5, U6) U1 snRNP Process involves catalytic activity of certain RNA species (RNA enzymes) also called “ribozymes” Splicing of pre-mRNA – the principle 1. Cleavage at 5′ splice site (SS) and joining of 5′ end of intron to an A within intron (branch point). Splicing of pre-mRNA – the principle 1. Cleavage at 5′ splice site (SS) and joining of 5′ end of intron to an A within intron (branch point). 2. Cleavage at the 3’ splice site and simultaneous ligation of exons, resulting in excision of intron http://youtu.be/aVgwr0QpYNE mRNA to protein: translation Components of translational process mRNA Components of translational process amino acyl tRNA mRNA Components of translational process amino acyl tRNA ribosome large unit mRNA ribosome small unit  tRNA structure Figure 7-28 Essential Cell Note: 70-80 nucleotides long, complementary base pairing (loops), Biology (© Garland Science modified bases (eg pseudouridine, dihydrouridine) 2010)  Linkage of amino acid to tRNA 1. amino acid eg alanine coupled with AMP catalysed by family of aminoacyl tRNA synthetases (@40 tRNA species/cell) Linkage of amino acid to tRNA 1. amino acid eg alanine coupled to AMP catalysed by family of aminoacyl tRNA synthetases (@40 tRNA species/cell) 2. aminoacyl AMP combines with tRNA to form aminoacyl tRNA Ribosome- a molecular ‘machine’ which interprets the mRNA code E.coli: @20,000 per cell (25% dry weight) Dividing mammalian cell: 1x 107/cell Ribosomal RNA structure/sequence E.coli 16S rRNA Structural scaffold for proteins Catalytic activity (eg peptidyl transferase of large su) Ribosomes consist of RNA and proteins green 50S protein (light green RNA) blue 30S protein (light blue RNA)  Ribosome structure small subunit dark green large subunit light green Each ribosome has a binding site for mRNA and three bindings sites for tRNA (E, P, A sites) Translation- overview next session: translation stages gene regulation  Key texts The following are all good texts that cover the same material. Most give you MUCH more detail: – Cooper, G.M. & Hausman, R.E. (2019). The cell, a molecular approach (8th ed.). Blackwells. http://www.ncbi.nlm.nih.gov/books/NBK9839/ (2nd Ed) – Lodish et al. (2012). Molecular Cell Biology, 7th Ed, Freeman; https://www.ncbi.nlm.nih.gov/books/NBK21475/ (4th Ed) – Alberts et al. (2015). Molecular Biology of the Cell (6th ed). Garland Science. http://www.ncbi.nlm.nih.gov/books/NBK21054/ (4th Ed)  Online resources Other animations and video tutorials DNA learning centre – Cold Spring Harbours online DNA learning resource - https://dnalc.cshl.edu/websites/ DNA from the beginning – An animated primer of 75 experiments that made modern genetics http://www.dnaftb.org/ Bozeman Science basic video tutorial, Transcription and translation http://youtu.be/h3b9ArupXZg