Gene Regulation Flashcards
7 ways to regulate protein concentration in a cell
- Control of synthesis of primary RNA transcript
- Post-transcriptional modifications of mRNA
- Degradation of mRNA
- Protein synthesis (translational control)
- Post-translational modification of protein
- Targeting and transport of the protein
- Degradation of the protein
- Effect the steady-state concentration of a protein
- Each process has several potential points of regulation
major processes regulating protein levels
- Cell protein concentration is controlled by:
o Gene transcription: regulation of the frequency of a mRNA synthesis
o mRNA degradation: rate at which that mRNA is degraded
o Protein translation: rate at which that mRNA is translated into protein
o Protein degradation: rate at which a protein is degraded
process to determine protein levels
- Mass spectrometry – to measure protein concentrations
- mRNA sequencing (RNA-seq) – to measure mRNA levels
- mRNA protection from ribonuclease digestion by associated ribosomes (ribosome footprinting) – to estimate translation rates
- Stable isotope labeling – to determine degradation rates
- Statistical analysis of the data – to correct for inherent biases and errors in these methods
overview of RNA processing and post-transcriptional gene control
- Pol I => tRNA
- Pol II => mRNA
- Pol III => rRNA
- Gene expression regulation
o Transcriptional regulation
o mRNA processing
o Cytoplasmic regulation - RNA processing
RNA processing
RNAs from protein-coding genes – transcribed by RNA polymerase II, processed from primary transcripts in the nucleus before export to the cytoplasm
Step 1 – Use of alternative exons during pre-mRNA splicing
Step 2 – Use of alternative poly(A) sites.
Step 3 – Properly processed mRNAs – exported to the cytoplasm, Improperly processed mRNAs –
• blocked from export to the cytoplasm
• degraded the exosome complex containing multiple ribonucleases
Step 4 – Translation initiation factors – bind to the 5′ cap cooperatively with poly(A)-binding protein I bound to the poly(A) tail and initiate translation
Step 5 – mRNA degraded in cytoplasmic P bodies – translational repression, Deadenylated and decapped by enzymes, Degraded by cytoplasmic exosomes, Control of mRNA degradation rate – regulates mRNA abundance and amount of protein translated
Step 6 – mRNAs synthesized without long poly(A) tails – translation regulated by controlled synthesis of a long poly(A) tail by a cytoplasmic poly(A) polymerase
Step 7 – Translation regulation by other mechanisms – miRNA (~22-nucleotide RNAs) – inhibit translation of mRNAs to which they hybridize, usually in the 3′ untranslated region
Step 8a – tRNAs – transcribed by Pol III and processed in the nucleus
Step 8b – rRNAs – transcribed by Pol I – processed in the nucleolus
Step 9 – Regions of precursors cleaved from the mature RNAs – degraded by nuclear exosomes
Eukaryotic gene expression step 1: transcriptional control
- Transcriptional control is regarded as the primary means of regulating gene expression
- Cis-acting control elements regulate transcription
- Promoters determine the transcription start site & direct binding of RNA pol II (eg TATA box in rapidly transcribed genes)
- Promoters are located near the start site
- Cis-acting elements - DNA sequences in the vicinity of the structural portion of a gene that are required for gene expression
- Other elements (enhancers and repressors) help regulate a particular gene
o these are often tissue specific and function only in specific differentiated cell types
o Enhancers and repressors elements may be many kb to 100s of kb away from the transcriptional start site
regulation of activators and repressors
Small-Molecule Effectors Can Regulate Activators and Repressors
- Repressors reduce RNA Pol-promoter interactions or block the polymerase.
o bind to operator sequences on DNA, usually near a promoter in bacteria but further away in many eukaryotes
- Effectors can bind to repressor and induce a conformational change.
o change may increase or decrease repressor’s affinity for the operator and thus may increase or decrease transcription
transcription factors (TF)
proteins which bind to regulatory elements of a gene - control transcription
- synthesised or activated in response to external stimuli (hormone binding)
- 2 categories: general or tissue/cell specific
- some genes expressed in all cells - called house-keeping genes, ie constitutive transcription
- other genes only expressed in certain tissues, involves tissue specific TF and promoters on the genes, ie regulated transcription
gene regulating terms
- housekeeping gene
- regulated gene
housekeeping: under constitutive expression, constantly express in approx all cells
regulated gene: levels of the gene change with the needs of the organism. genes are inducible (able to be turned on) and repressible (able to be turned off)
promoter: similar for all genes
regulatory sequence: different for all genes
Eukaryotic gene expression step 2: post transcriptional mRNA processing
- The initial transcripts are large and sequences represent a range of expressed genes depending on cell type - called heterogeneous mRNA (hnRNA).
- hnRNAs are capped at the 5’ end using GTP as substrate. This becomes a ribosome recognition site for initiation of translation.
- Poly A tail is added (plays a role in RNA degradation)
- mRNA is then spliced specifically for cell
eukaryotic genes are divided into introns and exons - Exon defined as the fragment of DNA which is
- represented in the mature mRNA.
- Note: an exon can be coding or non-coding
RNA splicing joins axons
- Splice site forms a loop with branch site.
- Ligation of the two exons frees the lariat (loop).
- Intron is degraded
- Splicing occurs in spliceosome- large RNA-protein complex of 5 snRNPs ‘snurps’
- snRNP-snRNA & proteins
constitutive vs alternate splicing
const: consecutive exons are kept
alt: only some exons are kept
troponin - alternative splicing
- one gene = 64 mRNAs
- Different troponins have different binding affinities for Ca++ and causes different rates of actin-myosin (muscle) contraction
- Some exons are constitutive
- Some exons are alternative (mutually exclusive)
- Some are combinatorial and used in and or fashion
- Control of splicing is complex.
- Without splicing would need for 64 different genes, with own promoters etc. Splicing is more efficient.
RNA transport & localisation
- Export of RNA is delayed until processed completely
- “left-over” / damaged RNA is degraded in exosomes - by exonucleases
- Some mRNAs are localised to specific regions …
- in the cytosol, RNA is bound by ribosomes and translated …or the ER - secreted proteins
- Advantage to make protein close to where it’s needed … 3’-UTR usually contains specific signals in the mRNA to direct this
- mRNAs also carry information to specify:
o half-life e.g. AU elements
o efficiency of translation (miR binding sites)
Eukaryotic gene expression step 3: mRNA degradation
- Half-life of eukaryotic mRNAs can be hours
- Many less stable (30 minutes or less)
- Specific sequences in mRNAs determine both the pathway and kinetics of degradation
- Generally begins with poly-A tail shortening by an exonuclease, which is slow
- then, de-capping followed by 5’->3’ degradation
- The proteins that catalyse tail shortening compete with translational proteins, both use the polyA tail & 5’ cap!
