SBP3411 Gene Expression in Eukaryotic Cells PDF Lecture Notes

SBP3411 Gene Expression in Eukaryotic Cells Dr. Hanis H. Harith Dept. of Biomedical Science, UPM [email protected]  Learning Outcomes At the end of this lecture the student is able to: Identify the role of different types of RNA molecules in eukaryotic gene expression D...

SBP3411 Gene Expression in Eukaryotic Cells Dr. Hanis H. Harith Dept. of Biomedical Science, UPM [email protected]  Learning Outcomes At the end of this lecture the student is able to: Identify the role of different types of RNA molecules in eukaryotic gene expression Describe the key events and molecules involved in RNA synthesis and processing in eukaryotic cells Discuss the role of transcription regulators in eukaryotic gene expression Describe examples of mechanisms of post-transcriptional control at different levels Hanis Harith SBP3411 (2023) Dept of Biomedical Science UPM Lecture Outline Types of RNA & RNA polymerases RNA synthesis and processing Transcriptional controls Post-transcriptional controls Hanis Harith SBP3411 (2023) Dept of Biomedical Science UPM  DNA organization in eukaryotic cells Storage of hereditary information All cell types in a multicellular organism contain the same DNA content Eukaryotic DNA is packaged into multiple chromosomes Chromosome packing occurs on multiple levels High level of DNA organization prevents the DNA from becoming tangled but still remain accessible to enzymes or proteins involve in DNA replication, DNA repair and gene expression Alberts et al. (2014). Essential Cell Biology (4th Ed) Hanis Harith SBP3411 (2023) Dept of Biomedical Science UPM Flow of genetic information Alberts et al. (2019). Essential Cell Biology (5th Ed) Hanis Harith SBP3411 (2023) Dept of Biomedical Science UPM Alberts et al. (2014). Essential Cell Biology (4th Ed) RNA transcripts or molecules RNA carries info from the DNA (gene) transcribed - same language but different chemical forms Sugar: ribose vs deoxyribose Base: uracil vs thymine Structure: single-stranded vs double-stranded RNA polymerase: An enzyme that unwinds DNA Some RNAs (non-mRNAs) helix & catalyzes the formation of phosphodiester have structural, regulatory bonds between nucleotides or catalytic roles RNA polymerase II RNA polymerase I (most) RNA polymerase III (some) RNA polymerase II RNA polymerase III RNA polymerase II Hanis Harith SBP3411 (2024) Dept of Biomedical Science UPM Alberts et al. (2019). Essential Cell Biology (5th Ed) RNA synthesis in eukaryotic cells: Initiation Requires assembly of general transcription factors (TFs) on the promoter Promoter: DNA sequences upstream of transcription start site (+1) orient RNA polymerase on the promoter of the correct DNA template strand and ensure that transcription begins in the correct direction actual order may vary with promoters TFIID binds to TATA box (located at -30) TATA box is commonly found on promoters used by RNA polymerase II Recognized by TBP, a subunit of TFIID Binding of TBP bends the DNA double helix Movie 7.4 Alberts et al. (2019). Essential Cell Biology (5th Ed) Local DNA distortion by TFIID allows adjacent binding of TFIIB (at -35) Hanis Harith SBP3411 (2023) Dept of Biomedical Science UPM RNA synthesis: Formation of transcription initiation complex Assembly of other general TFs TFIIH exposes the template strand by prying apart the double helix at transcription start site (energetically unfavourable reaction; coupled with ATP hydrolysis) Binding of RNA polymerase completes the formation of transcription initiation complex To begin transcription, RNA polymerase needs to be released from most general TFs Requires phosphorylation of polypeptide tail of RNA polymerase by TFIIH TFIID remains bound Elongation factors will load onto actively transcribing RNA polymerase Dissociated general TFs can initiate transcription Alberts et al. (2019). Essential Cell Biology (5th Ed) with another RNA polymerase Hanis Harith SBP3411 (2024) Dept of Biomedical Science UPM RNA synthesis: Elongation & termination RNA Polymerase moves along the DNA and unwinds DNA helix Elongation factors facilitate movement of RNA Polymerase through DNA that is packaged into nucleosomes (not shown) Polynucleotide synthesis is driven by ATP hydrolysis via the alternative route Incoming ribonucleoside triphosphates (ATP, CTP, UTP, and GTP) are high E intermediates that react with the 3’ end of RNA chain Hydrolysis of the pyrophosphate is highly favorable and drive the overall reaction in the direction of polynucleotide synthesis Alberts et al. (2019). Essential Cell Biology (5th Ed) Movie 7.3 When transcription ends, RNA polymerase tail is dephosphorylated by protein phosphatases and it is released from the DNA Hanis Harith SBP3411 (2024) Dept of Biomedical Science UPM  Eukaryotic RNA processing: RNA capping & polyadenylation RNA processing: RNA capping, splicing, polyadenylation occurs as the RNA is being synthesized Facilitated by enzymes present on the phosphorylated tail of RNA polymerase The formation of mRNA requires both capping and polyadenylation RNA capping: attachment of a guanine nt bearing a methyl group at 5’ end when RNA transcript is about 25 nts long Polyadenylation: 3’ end is trimmed & addition of repeated adenine nts to form poly-A tail on newly transcribed mRNA by 2 different enzymes Alberts et al. (2019). Essential Cell Biology (5th Edition) Significance of capping & polyadenylation Increase stability of mRNA Facilitate export to cytosol Recognized by protein-synthesis machinery (checkpoint) Eukaryotic mRNA molecule Hanis Harith SBP3411 (2024) Dept of Biomedical Science UPM Organization of eukaryotic genes and pre-mRNA Alberts et al. (2019). Essential Cell Biology (5th Edition) Exons: short protein-coding sequences which are scattered and represent only a small fraction of the total gene Introns: long non-coding intervening sequences commonly found in protein- coding eukaryotic genes Both exons and introns are transcribed into RNA transcript (pre-mRNA) RNA splicing removes introns and stitches exons together occurs after capping & usually before poly-A-tail is added RNA splicing and processing is required for pre-mRNAs to become functional mRNA which can be exported for protein translation Hanis Harith SBP3411 (2023) Dept of Biomedical Science UPM RNA splicing by spliceosome Movie 7.5 The core of spliceosome is composed of snRNAs & proteins (snRNPs) Assemble on the pre-mRNA as it is being synthesized snRNAs act as ribozymes that catalyze the splicing reactions and formation of covalent bonds between exons Successful splicing is marked by exon junction complexes snRNPs recognize splice-site sequences on pre-mRNA signal the beginning and the end of an intron snRNAs bind to splice-site sequences by complementary base-pairing Introns are spliced out by forming a lariat structure, which is then degraded in the nucleus Alberts et al. (2019). Essential Cell Biology (5th Edition) Hanis Harith SBP3411 (2024) Dept of Biomedical Science UPM mRNA export is highly selective Alberts et al. (2019). Essential Cell Biology (5th Edition) Only correctly processed mRNA are exported to cytosol bound to poly-A-binding proteins, a cap-binding complex and exon junction complexes that bind to appropriately spliced mRNA Mediated by the nuclear pore complexes – act as gates that control which macromolecules enter or leave the nucleus Waste RNAs remain in nucleus and are degraded and can be reused for transcription E.g. excised introns, broken RNAs, aberrantly spliced transcripts may be harmful if allowed to leave the nucleus Hanis Harith SBP3411 (2023) Dept of Biomedical Science UPM Gene expression is mainly controlled by transcription regulators Movie 8.1 Transcription regulators switch transcription on (activator) or off (repressor) (Homeodomain) Proteins that recognize specific regulatory DNA sequences and form specific non-covalent interactions Regulatory DNA sequence can integrate information from many signals (e.g. environmental changes) to determine the rate of gene expression Gene activation can occur at a distance Looping of intervening DNA sequence permits contact with the general TFs and RNA polymerase Interaction with the transcriptional The whole complex machinery may be forms a TIC at the facilitated by a large appropriate position on complex of proteins the promoter (Mediator) Repressor proteins inhibit transcription by preventing TIC assembly or block RNA polymerase from moving forward Alberts et al. (2019). Essential Cell Biology (5th Edition) Hanis Harith SBP3411 (2024) Dept of Biomedical Science UPM Transcription regulators can recruit chromatin–modifying proteins DNA packaging (e.g. nucleosomes) can physically block assembly of TIC on the promoter Chromatin structure needs to be modified to allow access to promoter Gene activators enhance efficiency of transcription initiation by recruiting: enzymes that covalently modify histone proteins e.g. HATs: to recruit proteins that promote transcription including general TFs chromatin-remodeling complexes: increase accessibility of nearby DNA e.g. TATA box Alberts et al. (2019). Essential Cell Biology (5th Edition) Gene repressors reduce the efficiency of transcription initiation by recruiting enzymes that modify histone proteins to inhibit transcription (e.g. HDACs) Hanis Harith SBP3411 (2023) Dept of Biomedical Science UPM Transcriptional control in a multicellular organism Distinct cell types have the same DNA but differ in the sets of genes/proteins expressed Modified from Alberts et al. (2019). Essential Cell Biology (5th Edition) Some proteins are commonly expressed across cell types (e.g. housekeeping proteins) Distinctive cell properties are determined by expression of specialized proteins Expression of eukaryotic genes are controlled by combinations of transcription regulators (Combinatorial control) which direct the assembly and formation of a complete TIC Multiple transcription regulators bind to respective regulatory DNA sequences (vary with genes) Regulatory DNA sequences can integrate information from various signals to determine the transcription rate at the right time, conditions, and in specific cell type A single protein can coordinate the expression of multiple genes; it completes the combination of factors required to activate or repress the gene Alberts et al. (2019). Essential Cell Biology Hanis Harith SBP3411 (2023) Dept of Biomedical Science UPM (5th Edition) Post-transcriptional controls in eukaryotic cells Critical for most eukaryotic genes to prevent synthesis of unnecessary genes Alberts et al. (2019). Essential Cell Biology (5th Edition) The mechanisms that operate after transcription initiation to fine-tune the expression of many genes/proteins e.g. Alternative RNA splicing The presence of binding sites for repressor proteins in the 5’UTR region to prevent ribosome access to mRNA Mechanisms to control mRNA degradation Hanis Harith SBP3411 (2023) Dept of Biomedical Science UPM Alternative splicing of RNA in eukaryotic cells 5’ cap Poly-A tail Alberts et al. (2019). Essential Cell Biology (5th Edition) Exons may be skipped or included by spliceosome, but their order in the DNA sequence is maintained Advantages: Allows the production of different mRNAs and proteins from one gene in specialized cell types Enables eukaryotes to further increase the coding potential of their genomes Hanis Harith SBP3411 (2023) Dept of Biomedical Science UPM Mechanisms of mRNA degradation control mRNA lifespan determines protein expression level a single mRNA can be translated into protein many times e.g. short-lived mRNAs results in low protein expression mRNA stability varies depending on the nucleotide sequence (e.g. 3’ UTR region) which may contain binding sites for proteins involved in RNA degradation mRNA are eventually degraded by RNases in the cytosol mRNA degradation can also be regulated by miRNAs miRNA forms RNA-induced silencing complex (RISC) in the cytosol and target specific mRNA with complementary sequences (by base-pairing) Target mRNA is rapidly degraded by RISC or sequestered and degraded by nucleases Hanis Harith SBP3411 (2023) Dept of Biomedical Science UPM Alberts et al. (2019). Essential Cell Biology (5th Edition)