Lecture 9 CSF Gene to Protein 2024.pdf

tRNA http://www.xvivo.net/illustration/ Recap Lecture 8….. Lecture 9: From Gene to Protein At the end of this lecture, you should be able to: 1. describe the sequence of events in protein synthesis: -the process of transcription, including its initiation -the processing of pre-mRNA to mature mRNA -t...

tRNA http://www.xvivo.net/illustration/ Recap Lecture 8….. Lecture 9: From Gene to Protein At the end of this lecture, you should be able to: 1. describe the sequence of events in protein synthesis: -the process of transcription, including its initiation -the processing of pre-mRNA to mature mRNA -the link between DNA sequence and protein sequence -the process of translation at the ribosome 2. discuss primary, secondary, tertiary and quaternary levels of protein structure and the relationship between structure and function of proteins Very brief, you will cover this in Lecture 14 3. understand the concept of vesicular transport and secretion of a protein What is gene expression? Gene expression: the process of going from DNA to a functional product The Central Dogma DNA RNA PROTEIN Genotype : an organism's hereditary information Phenotype : actual observable or physiological traits Our genotype and its interaction with the environment determines our phenotype Objective One Gene expression – where and what DNA (deoxyribonucleic acid) is the heritable material that is used to store and transmit information from generation to generation RNA (ribonucleic acid) acts as a messenger to allow the information stored in the DNA to be used to make proteins Proteins carry out cellular functions Three main steps: Transcription of RNA from DNA Processing of the pre-mRNA transcript Translation of the mRNA transcript to a protein you need to be able to sketch/label the above summary Campbells, 11th, 17.4 Objective One First step: Transcription – an overview Three steps: 1. Initiation 2. Elongation 3. Termination 1. Polymerase binds to promoter 2. moves downstream through the gene, transcribing RNA 3. detaches after terminator reached We focus on transcription of mRNA, not rRNA or tRNA, they use different polymerases DNA RNA uses the nitrogenous base Uracil, in place of Thymine and it is single stranded, while DNA is double stranded Objective One Transcription – initiation - a closer look Assembly of multiple proteins required before transcription can commence Also known as the “coding” strand TATA box typically ~25nt upstream the “non-coding” strand Assembly of several transcription factors including the TATA box binding protein (TBP) RNA Pol II can now bind along with more transcription factors to form the transcription initiation complex And so transcription begins..… (note, there are many additional initiation factors in this process) Objective One Transcription – elongation & termination - a closer look 10-20 nucleotides exposed at a time when DNA unwound Elongation: Complementary RNA nucleotides added to 3’ end of growing transcript (3’OH of transcript binds with 5’ phosphate of incoming nucleotide) It forms a phosphodiester bond Double helix reforms as transcript leaves the template strand Termination: after transcription of the polyadenylation signal (AAUAAA) nuclear enzymes release the pre-mRNA and RNA polymerase then dissociates from the DNA Fidelity (proofreading) is less than for DNA replication The pre-mRNA transcript is now ready for further processing Objective One Second step: mRNA processing -capping, tailing and splicing Capping: a modified guanine nucleotide is added to the 5’ end Tailing: 50-250 adenine nucleotides (polyA) are added to the 3’ end Why? Capping and tailing are thought to facilitate export, confer stability and facilitate ribosome binding in cytoplasm) Splicing: introns are removed from the transcript Definitions to know: Exons: regions that remain in mature RNA (includes UTR) UTR: untranslated regions at 5’ and 3’ ends of mRNA Introns: intervening regions that do not remain in mature RNA Objective One Where does splicing occur? At the spliceosome, within the nucleus Spliceosome: a large complex of proteins and small RNAs Introns are removed from the transcript and exons are rejoined to form mature mRNA Alternative splicing is a process by which different combinations of exons are joined together. This results in the production of multiple forms of mRNA from the same pre-mRNA population. Alternative splicing allows for multiple gene products from the same gene ~20,000 genes, there could be many times that number of proteins!! Objective One Genes and their products can vary greatly in size!!! Details not examinable, just examples of diversity in gene /protein size Two of my favorite genes…. TATA-box binding protein (TBP): gene spans 18,000 bp (base pairs) of chromosome 6 so pri-RNA is ~18,000 nt and mRNA is ~1900 nt (nucleotides) has 8 exons coding for a protein of 339 amino acids Huntingtin (HTT): gene spans 180,000 bp of chromosome 4 has 67 exons coding for a protein of 3144 amino acids the human genome ~3000 Mbp (million base pairs) ~20,000 genes Objectives 1 to 4 United by sequence protein sequence determines its final structure structure determines function DNA mutations can affect ability of the protein to function You need to be able to use these codon tables, not memorise them! Objective One Third step: Translation – an overview Mature mRNA transcript exits nucleus and is bound by the ribosome Codons are translated into amino acids tRNA molecules within the cytosol with specific anticodons carry corresponding amino acids Hydrogen bonds form between mRNA and anticodon of the appropriate tRNA The amino acid is added via peptide bonds to the growing polypeptide chain Three main steps: initiation, elongation, termination Objective One Ribosome has binding sites for mRNA and tRNA tRNA and mRNA held within ribosome to enable the formation of the polypeptide mRNA binding site on small subunit A site: holds “next-in-line” tRNA P site: holds tRNA carrying the growing polypeptide E site: tRNAs exit from here Q: How do the new amino acids arrive? Objective One Transfer RNA (tRNAs) – the translator tRNA is the physical link between the mRNA and the amino acid sequence of proteins Objective One Translation - initiation – a closer look Initiator tRNA = tRNA carrying methionine (Met) Small ribosomal subunit with initiator tRNA already bound binds 5’ cap of mRNA Small ribosomal subunit scans downstream to find translation start site (AUG) Hydrogen bonds form between initiator anticodon and mRNA Large ribosomal subunit then binds – completing the initiation complex Energy (GTP- Guanosine triphosphate) is required for assembly many additional initiation factors are required Note: Tortora and this image don’t show it, but in eukaryotes, the initiator is already bound to the small subunit before mRNA binds Objective One Translation - elongation – a closer look Codon recognition: base pairs with complementary anticodon GTP invested to increase accuracy / efficiency Peptide bond formation: A large subunit rRNA catalyses peptide bond formation Removes it from tRNA in P site Translocation: moves tRNA from A to P site tRNA in P site moves to E and is released Energy is required (GTP) Empty tRNAs are ‘reloaded’ in the cytoplasm using aminoacyl-tRNA synthetases additional elongation factors are required Objective One Translation - termination – a closer look mRNA stop codon in the A site is bound by a release factor additional termination factors are required Bond between p-site tRNA and last amino acid is hydrolysed, releasing polypeptide Hydrolysis of two GTP molecules required Ribosome components can be recycled Objective One Gene expression is tightly regulated Multiple control points: Transcription factors need to assemble, and DNA needs to be accessible capping, extent of polyadenylation, alternate splicing, producing an mRNA able to be translated specific proteins assist in nuclear export of mRNA Campbells, 11th, 17.25 regulatory proteins can block translation, variable mRNA life-spans Objective One Why is control of gene expression important? To achieve the right thing at the right time in the right place!! (this is temporal and spatial control) Lecture 8 cell signaling Housekeeping (commonly used) proteins are continuously produced protein and mRNA are present in large quantities (e.g. Tubulin) Microtubules, Lecture 6 typically, have longer “half life” in cells Other proteins are produced in response to stimuli as required cell signaling (e.g. ligand binding a cell surface receptor, or activating an intracellular receptor) signal transduced and may enter nucleus to activate transcription results in the production of a short-lived protein to carry out the required function Aim to do some revision and then be able to label this without needing your notes at all, see page 78 of lecture guide Objective Two Amino acid properties The side chains (R groups) determine the properties of each amino acid You do not need to know all of the amino acid properties for 107, but you do need to appreciate that they collectively determine the final structure and function of the protein There are twenty standard (coded for) amino acids Objective Two Primary Structure N-terminus Protein sequence (primary structure) is determined by DNA sequence You need to know which end is N and C DNA and RNA is read 5’ to 3’ Held by covalent bonds between amino acids (relatively strong) N is to 5’ as C is to 3’ The polypeptide starts to form secondary structures as soon as it leaves the ribosome C-terminus Objective Two Secondary, tertiary and quaternary structures Held by weak hydrogen bonds to form alpha helix and beta sheets More detail in Lecture 14 with Michael 3D shape stabilized by side chain interactions Multiple proteins associate together to form a functional protein Not all proteins form quaternary structures Objective Three Protein processing and sorting Remember the endomembrane system, Lecture 6? All translation commences on free ribosomes Many proteins are processed and sorted through the RER and Golgi – but not all Proteins destined to function in the cytosol – complete translation on free ribosomes Proteins that go through the endomembrane system complete translation at fixed ribosomes on the RER Q: how does a ribosome “know”? Objective Three Signal peptides direct ribosomes to RER Signal peptide: at N terminus of the protein (~20aa) SRP: signal recognition particle At step 6: A secretory protein such as insulin is solubilized in lumen, while a membrane protein remains anchored to the membrane Both then go to the Golgi via vesicles for further maturation Objectives 3&4 Post‐translational modifications Translation is now complete, but the protein may not yet be functional Common (there are 100s) post‐translational modifications include: Phosphorylation (addition of a phosphate group) Methylation (addition of a methyl group) Acetylation (addition of an acetyl group) Biotinylation (addition of biotin) Carboxylation (addition of a carboxylic acid group) Carbohydrate addition (particularly for membrane bound proteins, eg. glycoproteins) Cleavage Ubiquitination Some occur within the Golgi, others in the cytosol (eg. Phosphorylation (Lecture 8)) Can confer activity – eg. via phosphorylation or enzyme cleavage or ability to interact with other molecules – eg. biotinylation, methylation of histones or direct to particular locations – eg. ubiquitination for proteasome degradation Some useful resources for this lecture protein synthesis overview, 2:41m https://www.youtube.com/watch?v=gG7uCskUOrA Also on module page https://www.youtube.com/watch?v=8kK2zwjRV0M Is ~12 minutes, useful background stuff, not examinable If you want more info on 5’ – 3’ and N and C terminus: https://www.youtube.com/watch?v=Rn9zldxtZko (not examinable, just helpful) Complete the image on page 70 as part of your revision Publisher permission was granted for lecture slide use of images/resources from the 107 texts (Tortora and Campbell). Unless otherwise stated content was sourced from these texts or were lecturers own Next CSF Lecture : Cell division Tip: please try to watch this mitosis video (also on my Canvas module) BEFORE the next lecture