Gene Expression in Eukaryotic Cells

Gene Expression in Eukaryotic Cells

• This lecture covers gene expression in eukaryotic cells
• Students will learn about the role of different RNA molecules in eukaryotic gene expression
• They will also learn about the key events and molecules involved in RNA synthesis and processing.
• The role of transcription regulators in eukaryotic gene expression will be discussed.
• Examples of post-transcriptional control mechanisms at various levels will be presented.

Lecture Outline

• Types of RNA & RNA polymerases: Details about different types of RNA and their functions. RNA polymerases and their roles will be covered.
• RNA synthesis and processing: This section will cover RNA synthesis and processing in eukaryotic cells.
• Transcriptional controls: This will cover the details on how transcription is controlled in eukaryotic cells.
• Post-transcriptional controls: Details of post-transcriptional control mechanisms in eukaryotic cells.

DNA Organization in Eukaryotic Cells

• DNA stores hereditary information
• All cells in a multicellular organism have the same DNA content.
• Eukaryotic DNA is packaged into multiple chromosomes.
• Chromosome packing occurs on multiple levels, compacting the DNA significantly.
• High levels of DNA organization prevent entanglement while keeping DNA accessible for replication, repair, and gene expression.

Flow of Genetic Information

• Genetic information flows from DNA to RNA (transcription) and then to protein (translation).
• DNA contains the instructions for making proteins.
• Transcription copies the DNA code into RNA.
• Translation translates the RNA code into a protein.
• RNA molecules play important roles in this process.
• DNA can be copied or replicated.
• The segments of DNA that are transcribed to RNA are called genes (orange).

RNA Transcripts or Molecules

• RNA carries genetic information from DNA, utilizing different chemical forms (e.g., ribose vs. deoxyribose).
• RNA is single-stranded, unlike its DNA counterpart (in most cases).
• RNA polymerase unwinds DNA to catalyze nucleotide joining.
• Different types of RNA have specific functions in cells. (e.g., messenger RNA, ribosomal RNA, transfer RNA...).

RNA Synthesis in Eukaryotic Cells: Initiation

• General transcription factors (TFs) assemble on the promoter region.
• The promoter is a DNA sequence upstream of the gene.
• RNA polymerase II is oriented on the promoter to start transcription in the correct direction.
• The order of TF binding can vary between promoters.
• TFIID binds to the TATA box, which is frequently found upstream of promoters used by RNA polymerase II.
• Adjacent binding of TFIIB occurs due to DNA distortion caused by TFIID.

RNA Synthesis: Formation of Transcription Initiation Complex

• General TFs are assembled to initiate transcription.
• TFIIH opens the DNA helix at the transcription start site.
• RNA polymerase II binding completes the initiation complex.
• RNA polymerase II must be released from general TFs to begin transcription.
• TFIIH phosphorylates the RNA polymerase II tail. -Dissociated general TFs can initiate transcription with other RNA polymerases.

RNA Synthesis: Elongation & Termination

• RNA polymerase moves along DNA to unwind the DNA helix.
• Polynucleotide synthesis is driven by ATP hydrolysis.
• Incoming ribonucleotides are added to the 3' end of the growing RNA chain.
• Pyrophosphate hydrolysis drives the overall reaction.
• The RNA polymerase tail is dephosphorylated to release the enzyme from the DNA.

Eukaryotic RNA Processing: RNA Capping & Polyadenylation

• RNA processing occurs as RNA is synthesized.
• RNA capping involves adding a modified guanine nucleotide to the 5' end.
• Polyadenylation adds a poly(A) tail to the 3' end.
• These processes increase mRNA stability and facilitate export from the nucleus.
• Enzymes are present on the phosphorylated tail of RNA polymerase that facilitate these changes.

Organization of Eukaryotic Genes and pre-mRNA

• Exons are the coding sections of genes (fragments).
• Introns are non-coding sections of genes that are removed in RNA processing.
• RNA splicing removes introns and joins exons.
• This occurs after capping but before polyA tail addition.
• Spliced pre-mRNA is ready to be translated.

RNA Splicing by Spliceosome

• Spliceosomes are composed of snRNAs and proteins.
• snRNAs catalyze splicing reactions and form bonds between exons.
• Exons are stitched together after the introns are removed.
• Introns are either degraded or used in other cellular processes.

mRNA Export

• Correctly processed mRNA is exported from the nucleus to the cytosol.
• This is facilitated by proteins that bind to mRNA.
• Nuclear pores facilitate mRNA transport.
• Unprocessed mRNA and other RNAs are degraded in the nucleus.

Gene Expression is Mainly Controlled by Transcription Regulators

• Transcription regulators switch transcription 'on' (activators) or 'off' (repressors).
• These proteins bind to specific DNA sequences.
• Gene activation can occur at long distances through DNA looping.
• Repressors prevent transcription initiation.

Transcription Regulators Recruit Chromatin-Modifying Proteins

• DNA packaging (nucleosomes) can block initiation.
• Proteins modify chromatin structure to allow access.
• Activator proteins enhance transcription initiation via recruitment of chromatin-modifying enzymes (e.g., HATs).
• Repressor proteins hinder transcription initiation ( e.g., HDACS).

Transcriptional Control in a Multicellular Organism

• Cells in a multicellular organism express unique sets of genes, determined by transcription regulators.
• Combined action of different transcription factors leads to specific changes in transcription rates.
• Regulatory DNA sequences integrate signals to activate or repress transcription.
• Certain genes are constitutively present across all cell types.

Post-transcriptional Controls in Eukaryotic Cells

• Mechanisms that regulate gene expression after initiation.
• Alternative splicing, mRNA degradation, and translation control.
• mRNA stability varies based on nucleotide sequences that may or may not contain binding sites for degradation proteins.
• Proteins modify mRNA, potentially preventing translation or initiating degradation.
• miRNA- mediated mechanisms are present, affecting mRNA degradation.

Alternative Splicing of RNA in Eukaryotic Cells

• Exons can be skipped or included in mRNA, leading to multiple mRNAs from a single gene.
• This allows for a larger variety of proteins from a limited DNA sequence.

Mechanisms of mRNA Degradation Control

• mRNA lifetime determines protein levels.
• Short-lived mRNA usually results in lower protein levels.
• mRNA with sequences containing binding sites for proteins that facilitate degradation are destroyed.
• Degradation controlled by specific proteins or via mRNA modification.