BCHS317 Study unit 2: Transcription and translation in eukaryotic cells Dr Fortunate Mokoena Fortunate.Mokoena@nwu.ac.za SU2 Study Unit Outcomes 1. Eukaryotic gene promoters 2. Eukaryotic gene structure 3. Transcription elements 4. Transcription factors 5. Tissue specific gene expression 6. Initiation of protein synthesis and regulation 7. Prokaryotic vs eukaryotic protein synthesis 8. Post-translation processing of polypeptides Initiation of transcription GG: Ch 29 p: 1062 - 1064 Nucleosome structure End result with respect to transcription: DNA is compacted and inaccessible to transcriptional machinery Core histones • The core octamer structure consists of 2 copies each of the four core histone proteins: H2A, H2B, H3 and H4. • Nucleosomes: 147 bp of the DNA helix is wound 1.6 times around these core octamers • Histone H1 adds 29 – 43 bp of DNA to link consecutive nucleosomes. • H1 binds on outside to stabilize the complex Core histones Kim, Y. Z. (2014). Altered Histone Modifications in Gliomas. Brain Tumor Research and Treatment, 2(1), 7–21. http://doi.org/10.14791/btrt.2014.2.1.7 B&D Fig 32. 4 •N terminal tails of H3 and H4 accessible on surface of nucleosome. •Tails have Lys and Ser residues •Residues can be modified through lysine acetylation, lysine methylation, serine phosphorylation, lysine ubiquitination, and lysine sumoylation (addition of SUMOs - small ubiquitin-like modifiers). •Modifications impact on chromatin condensation and gene expression Figure 29.30 Diagram of possible modifications on the histone tails in the nucleosome. acK = acetylated lysine residue; meK = methylated lysine residue; meR = methylated arginine residue; PS = phosphorylated serine residue. • For the activation of transcription: - There has to be relief from repression caused by the compacted chromatin structure - RNA pol II must be able to interact with the promoter and transcription regulatory proteins • Activation of transcription occurs when the following factors have been met: (i) (ii) Recognition of the chromatin Alteration of the nucleosomes • When the nucleosome structure is disrupted, the DNA molecule is relaxed and the promoter region accessible for transcription • NB!!! The promoter is a segment on the DNA that RNAP and transcription regulatory proteins binds to initiate transcription of a particular gene. If the promoter is not accessible, the transcription cannot take place • In order for transcription to occur, the following factors are important: - Chromatin-remodeling complexes: mediate ATP-dependent conformational changes in nucleosome structure - Histone-modifying enzymes: introduce covalent modifications into the N-terminal tails of the histone core octamer • Chromatin remodeling and histone modification are closely linked processes Chromatin remodelling complex • Chromatin-remodeling complexes are large nucleic-acid –stimulated multi subunit ATPases - 1 megadalton • Introduce ATP dependent conformational (noncovalent) changes to nucleosome structure • Loosen DNA-protein interactions in the nucleosomes (slide, eject, insert or restructure core octamers) • 50 bp of DNA “peels” away from the nucleosome • This creates a bulge which enables pol II and GTFs to bind • FACT (facilitates chromatin transcription) is a heterodimer that acts on some nucleosomes to catalyze H2A/H2B removal and re-addition • FACT activity is increased by ubiquitination of H2B Lys120 Histone modifying enzymes • Histone modification enzymes introduce covalent modifications into the N terminal tails of the core octamer - side chains on histones are modified • Thus, DNA-histone associations are diminished How? 1. Initially during transcriptional activation, there is acetyl-CoA-dependent acetylation of amino groups on lysine residues in the histone tails via histone acetyltransferases (HATs) - Acetylation suppresses positive charge in the histone tails electrostatic interactions between DNA and histones are disrupted - Histone deacetylase complexes remove acetyl groups from lysine residues favors chromatin compaction 2. Modifications to the histone tails recruit binding of new protein participants - E.g. Creates binding sites the chromatin remodeling complexes. Acetylation of histones enhances access to promoter region and facilitates transcription. Examples of histone modifications as well as the effect it has Eukaryotic gene activation • Initiation of transcription is specialized and tightly controlled to ensure that the correct genes are transcribed in the correct quantities • Results in a cascade of events in which different transcription elements and factors are involved • Transcription initiation in eukaryotes proceeds through assembly of a preinitiation complex (PIC) • The PIC consists of: - RNA polymerase II - A set of general transcription factors (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, and TFIIS) - A complex called Mediator (Srb/Med) - The CTD of pol II anchors Mediator - Mediator allows Pol II to communicate with transcriptional activators bound at sites distant from the promoter Example of tissue specific gene expression and multiple regulation of the same gene Response elements • Other regulatory elements include response elements • Found near the promoter and can bind specific TF • Proteins that trigger expression of related genes in response to environmental factors such as heat shock or hormones • Examples include - the heat shock element (HSE) - the glucocorticoid response element (GRE), and - the metal response element (MRE) Response elements Figure 29.27. Metallothionein binds metals and protects the cell agains heavy metal poisoning. The metallothionein gene possesses several constitutive elements in its promoter (the TATA and GC boxes) as well as specific response elements such as MREs and a GRE. The BLEs are elements involved in basal level expression (constitutive expression). TRE is a tumor response element activated in the presence of tumorpromoting phorbol esters such as TPA (tetradecanoyl phorbol acetate).
