L2: Gene Expression Basic Processes Flashcards
biological relevance of gene expression and regulation
- underlies many fundamental processes
- development
- cellular function
- organismal diversity
- human variation
- personalized medicine
- genetic disease
- cancer
therapeutic relevance of gene expression and regulation
- many therapies and biomedical technologies require understanding and manipulating of gene expression
- gene therapy
- recombinant DNA research
- production of biological therapeutics
- RNAi therapies
- CRISPR/Cas9
- Systems biology
- IPSCs
genetic differences in drug responsiveness
- lie in non-coding regions
- may affect expression of nearby genes
DNA -> RNA
- mRNA transcribed from template strand with complementary sequence
- occurs 5’ - 3’ in opposite orientation to template strand
where does RNA polymerase bind
- binds to the promoter
- elongation with RNA pol moving down DNA adding nucleotides to the 3’ end of the transcript
prokaryotic (bacterial) gene expression (and how different from eukaryotic)
- transcription and translation occur in same cellular compartment because there is no nucleus
- processes occur simultaneously
- no RNA splicing
- mRNAs are commonly polycistronic - multiple coding sequences to make different proteins
- multiple proteins from same transcript
- all genes transcribed by single RNA polymerase
- bacterial-induced disease
eukaryotic gene expression (and how different from bacterial)
- multiple compartments
- genes are monocistronic
- genes have introns and exons
- RNA splicing to eliminate introns
- different classes of genes (tRNA, rRNA, mRNA) transcribed by distinct RNA polymerases
- DNA packaged into chromatin
where eukaryotic transcription and RNA processing occurs
- nucleus
where eukaryotic translation occurs
- cytoplasm
exons
- coding and untranslated regions
RNA Pol I transcribes
- rRNA genes
- 28S
- 18 S
- 5.8S
RNA Pol II transcribes
- mRNA genes
- also microRNAs (regulation)
- snRNAs (RNA splicing)
RNA Pol III transcribes
- tRNA, 5S rRNA, additional small RNAs
- smaller genes
Are all three polymerases found in eukaryotes?
- yes found in all eukaryotes
Each RNA polymerase recognizes a different promoter that differs n sequence
what is the 4th RNA polymerase?
- mitochondria
- have their own
RNA polymerase II promoters
- where RNA polymerase and transcription starts
core promoters
- multiple types and elements
important class of core promoter
- TATA box - TATAA sequence
- 25 bp upstream of promoter
- binds TFIID
- initiator element (Inr) +1
TFIID
- TATA binding protein
- TBP + TAFs
enhancers
- bind TFs
- promote spatial, temporal, and quantitative control of transcription
upstream promoter-proximal elements
- GC rich regions - binds SP1 TF
- CAAT box - binds C/EBP
- regulate RNA levels
- bind transcription factors
- close to promoter (within 100 bp)
- elevate the levels of transcription
focused promoters
- focused at one site
- defined location
- contain TATA box and Inr elements
- regulated genes
- 20% of human promoters
dispersed promoters
- multiple start sites
- lie within CpG islands instead of TATA boxes
- housekeeping genes
- 80% of human promoters
- GC rich regions
housekeeping genes
- things that are pretty much on all the time.
- regulation of levels doesn’t change very much
regulated genes
- gene that gets turned on at specific times
RNA polymerase II composed of
- core enzyme composed of 12 subunits
General (Basal) transcription factors
- work with RNA polymerase to start eukaryotic transcription
- TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH
TATA binding factor
- TBF
- TBP-Associated Proteins (TAFs) - 13 proteins
TATA binding protein (TBP)
- part of TFIID
- binds TATA box
role of TFIIA and TFIIB
- stabilize TBP binding
- TFIIB positions and recruits RNA polymerase II over promoter
TFIIF role
- assists polymerase II binding
- followed by TFIIE and TFIIH
TFIIH role
- helicase
- unwinds DNA to open up helix and allow transcription to occur
- uses energy of ATP hydrolysis
TFIIH kinase role
- phosphorylates the C-terminal domain of polymerase II along with TFIIE
- RNA polymerase will not transcribe genes until phosphorylation occurs
- then transcription starts
which transcription factors are not released once transcription begins?
- TBP (remains at promoter to start another round of transcription)
- TFIIF (remains with Pol II)
First step of transcription initiation
- TFIID binds to TATA box
- because it contains TBP
- induces bending of DNA
second step of transcription initiation
- binding of TFIID is followed by binding of TFIIA and TFIIB
- stabilize TBP binding
- TFIIB positions and recruits RNA polymerase II over promoter at CRE and recruits TFIID
third step of transcription initiation
- RNA pol II recruited along with TFIIF
fourth step of transcription initiation
- TFIIE and TFIIF are recruited to form transcription initiation complex
what promoter does Pol I use?
- rlnr (ribosomal initiator) plus UPE/UCE sequences
- upstream promoter element
what promoter does Pol II use?
- TATA plus Inr (initiator)
- Inr plus DPE
- downstream promoter element
what promoter does Pol III use?
- internal sites (A and C for rRNA)
- A and B for tRNA
- has sequence elements that interact with pol III but are downstream of the genes
5S rRNA genes
- TFIIA and TFIIC recognize the promoter
tRNA gene
- TFIIC recognizes the promoter
other gene promoters
- use upstream TATA and PSE
- TATA recognized by TBP
related TBP-like proteins recognized by
- Pol I and Pol III
alpha amanitin toxin
- poisonous mushroom
- binds to RNA pol II and inhibits transcription
- causes severe kidney damage and can lead to death
prokaryotic transcription
- only one RNA polymerase for mRNA, tRNA, and rRNA genes
what does rifampin do?
- binds to the beta subunit of RNA pol and prevents initiation
- does not bind to eukaryotic RNA polymerases which is why we can use it on humans
prokaryotic promoters
- have two short conserved sequences (-35, -10) that are recognized by RNA polymerase
- looks like TATA box
prokaryotic RNA polymerase structure
- 5 subunits
- 2 alpha
- beta
- beta prime
- omega
sigma factors
- can vary
- allow holoenzyme (core + sigma) to recognize promoter
- allow it to target different promoters
leader
- 5’ untranslated region
- part of mRNA
trailer
- 3’ untranslated region
- has fairly extensive poly(A) tail that gets added on after the gene is transcribed
where do the untranslated regions come from
- exons that get spliced together
where does processing and modification of hnRNA occur?
- in the nucleus
hnRNPs
- interact with hnRNA to facilitate protein interactions, transport, and prevent degradation
hnRNA capped where?
- 5’ end
hnRNA poly(A)’d where?
- 3’ end
where does the transcript go after it is processed?
- the cytoplasm
5’ cap
- 7-methyl-guanosine
- first nucleotide that gets made
- odd 5’ to 5’ linkage using guanosine with a methyl group at N terminal (position 7) and position 2 of terminal ribose
function of 5’ cap
- makes mRNA resistant to degradation and enhances initiation of translation
when does capping occur?
- as mRNA is being transcribed
how are methyl groups donated?
- donated by SAM
- SAM regenerated after it donates a methyl group by vitamins B12 and folate (B9)
role of RNA pol in capping
- as Pol begins transcription, CTD phosphorylated by TFIIH kinase at Ser 5 binds capping proteins
- facilitates binding of cap proteins to CTD
- capping proteins transferred to newly emerged 5’ end of mRNA and cap the mRNA
- capping proteins put on the 7-methyl G
polyadenylation
- mRNA cleaved downstream at 3’ end and poly(A) tail added
- AAUAAA recognition sequence
is poly(A) present in the gene?
- no
- added by poly(A) polymerase with the use of ATP
what happens once poly(A) is absent?
- shortens over time
- mRNA degraded after absent
- way to control how long mRNA lasts
function of poly (A)
- mRNA stability
- transport mRNA from nucleus
- enhance translation
how does polyadenylation occur?
- cleavage specificity factors carried on the CTD of RNA pol II bind to the poly(A) signal sequence
- cleavage factors (CPSF, CstF) cut 3’ end
- Poly(A) polymerase recognizes 3’ end
- aided by CPSF begins synthesizing poly(A) tail
- Poly(A) binding protein binds to poly A stretch and directs extension of the sequence
- enhances ability of poly(A) to add more proteins
RNA splicing
- removal of introns and joining together of exons to create mature mRNA
splicing carried out by
- spliceosome
what recruits splicing factors?
- CTD of Pol II
snRNPs
- small nuclear RNA + proteins
- consists of the small RNAs: U1, U2, U4, U6 and associated proteins
precursor RNA
- specific sequences at 5’ splice acceptor site and 3’ splice donor site + branch site present in the intron
conserved sequences at intron-exon junctions
- recognized by spliceosome
- introns almost always start with GU 3’ site and end with AG at 5’ site
- A at branch site
splicing process
- U1 snRNP binds near first exon-intron junction at 5’ splice site after recognition of it
- U2 binds to conserved A residue in intron (at branch point sequence)
- snRNPs U4, U5, and U6 bind to U1-U2 complex and form a loop
- G at 5’ end of intron is cut and forms a 2’-5’ linkage with A residue at the branch site to form a lariat
- U1 and U4 released. U5 and U6 shift positions
- second cleavage occurs at 3’ end of intron after AG
- exons joined together, intron released along with the remaining parts of the spliceosome, and degraded
- part of spliceosome ligates the exons together
what percent of genetic diseases are due to splice mutations?
- 15%
Limb girdle muscular dystrophy symptoms
- weakness and wasting of muscles
gene for limb girdle muscular dystrophy
- LMNA
- codes for laming A and C which are intermediate filaments that support the nuclear envelop
what happens with limb girdle muscular dystrophy
- exon 8 gets spliced to exon 9
- change in G to C and exon 9 not spliced to exon 10
- results in truncated protein and RNA turnover
lupus cause
- autoimmune disorder
- cause is self-antibodies generated against splicing RNPs
- anti-smith antibodies binds to U1, U2, U4, U5, and U6 snRNPs
- less than 1% present in healthy individuals
anti-nRNP
- anti-U1NRP
- present in 30-40% of lupus patients and interact with U1snRNP
mitochondria genome
- distinct genome with rRNA, tRNA, and mRNA genes
- make own rRNA and tRNA
- genome is circular and small
mRNAs in mitochondria
- encode enzymes involved in electron transport and oxidative phosphorylation
what about transcription that takes place in mitochondria?
- they are all nuclear encoded genes
how to mitochondria get most of their proteins?
- they import them
- transcribed in nucleus, translated in cytoplasm, and imported into mitochondrion
- imported by TOM and TIM
70S subunit of prokaryotes composed of
- 50S and 30S
50S composed of
- 5S
- 23S +34 proteins
30S composed of
- 16S + 21 proteins
80S of eukaryotes composed of
- 40S and 60S
60S composed of
- 5S
- 5.8S on 28S + 50 proteins
40S composed of
- 18S + 33 proteins
mitochondria subunit
- 55S
55S subunit composed of
- 39S
- 28S
mitochondria rRNAs
- 16S
- 12 S
transcription and post-transcriptional cleavage of rRNA
- occurs in nucleolus
- 45S precursor rRNA (pre-rRNA) is transcribed by RNA polymerase I and associates with ribosomal proteins
- 5S RNA is a distinct gene cluster (~100 copies on chromosome 1) and transcribed by RNA polymerase III
- RNA is methylated and pseudouridylated and 45S RNA trimmed to a 41S precursor
- 41S with 5S cleaved to 32S with 5S and 20S
- 32S cleaved to 28S, 5.8S and 5S comes along
- 20S cleaved to 18S
- Cleaved into 28S, 18S, and 5.8S by ribonucleases
cleavage of 45S rRNA precursor
- ribonucleases cleave 45S into intermediate forms then eventually 28S, 18S, and 5.8S rRNAs
- 5.8S is hydrogen bonded with 28S
- snRNAs pair with rRNA precursors and guide location of modification and cleavage enzymes
- cleave the ends off the spacer sequences
- occurs in nucleolus
function of tRNAs
- adapter molecule that recognizes mRNA triplet code and transfers an amino acid to the growing polypeptide
features of tRNAs
- heavily modified bases
- dihydrouridine
- ribothymidine
- pseudouridine
- loops
- cloverleaf shape with defined loops
tRNAs of mitochondria
- mitochondria have their own tRNAs
3’ end of tRNA
- CCA where the amino acid gets attached
transcription of tRNA
- tRNA transcribed by RNA polymerase III from internal promoter
post transcriptional processing of tRNA
- done by nucleases
- 5’ and 3’ ends are trimmed
- small intron is removed and adjacent sequences are spliced (distinct from mRNA splicing)
- certain bases are modified by snoRNAs and modeling enzymes
nucleotidyltransferases
- replaces Us at the 3’ end and adds CCA
- CCA not encoded within the genome
highly repetitive DNA
- clustered at centromeres and telomeres - implicated in centromere function and chromosome pairing
moderately repetitive DNA
- some are transposable elements are defective transposable elements
- some are transcribed and produce functional RNAs needed in multiple copies
All repeats and LINEs
- transposons or remnants of transposons
energy of translation
- GTP and ATP
wobble position
- wobble at the third position
- one way for a tRNA that carries a specific amino acid to recognize a few different codons
- because of this, we don’t need a tRNA for every codon
aminoacyl-tRNA-synthetase
- recognize distinct amino acids and join them to tRNAs
- have proofreading capabilities and can tell when they have the wrong amino acid
aminoactyl-tRNA-synthetase reaction
- tRNA is charged covalently with an amino acid on CCA stem
- requires ATP
- yields aminoacyl-tRNA
- syntheses recognize acceptor stem and anticodon site
initiation of translation
- starts at AUG codon that codes for methionine
- eIF2-GTP delivers Met-tRNA to the 40S subunit connected with eIF3
- tRNA-Met is the only tRNA that can bind to the isolated 40S subunit - connected with GTP
- Cap at 5’ end of mRNA binds Cap-binding complex which recruits the preinitiation complex to mRNA
- mRNA binds to 40S subunit
- eiF4 scans mRNA and finds AUG codon (requires ATP)
- hydrolysis of GTP and eIF factors released
- large 60 S subunit binds to form functional ribosome
purpose of 40S subunit on mRNA
- connects with eIF3
- blocks premature binding of 60S subunit
cap binding complex composed of
- eIF4
binding sites
- E - ejection
- P - peptidyl
- A - aminoacyl-tRNA
met-tRNA binds where
- in the P site in the whole ribosome
- only tRNA that does this.
elongation of translation
- mRNA codon in A site determines which aminoacyl-tRNA binds
- new aminoacyl-tRNA binds elongation factor eEF1A-GTP
- complex binds to the A site, GTP hydrolyzed, and eEF1A-GDP is released
peptide bond formation
- in first round, AA in A site forms a peptide bond with the methionine in the P site
- amino group of aminoacyl-tRNA attacks carbonyl group of ester linkage of peptidyl-tRNA
- reaction catalyzed by peptidyltransferase which requires no energy source
peptidyltransferase
- not a protein but is the rRNA in the 60S subunit
- part of the 28S subunit
translocation of translation
- eEF2 complexes with GTP and binds ribosome
- makes space in the A site
- causes a conformational change that moves the mRNA and tRNAs with respect to the ribosome
- uncharged tRNA moves from the P site to the E site and is released
- peptidyl-tRNA moves to the P site.
- next codon occupies A site
- GTP hydrolyzed
termination of translation
- when a stop codon is reached, no aminoacyl-tRNA occupies the A site
- release factors bind to the ribosome and peptidyltranferase cleaves the peptide chain from the tRNA and protein is released
recycling of eEF1A
- important for continuous rounds of translation and translational regulation
- GTP hydrolyzed to GDP + Pi and eEF1A binds to eEF1Ba
- eEF1Ba exchanges GDP for GTP on eEF1A
- eEF1A is ready for the next round of translation
polysomes
- mRNAs are often translated by multiple ribosome, each generating a protein
protein targeting to sub cellular locations and transport
- proteins have targeting sequences that allow their transport to sub cellular destinations like the Golgi, ER, lysosomes, secretion at membrane
- proteins have a sequence at the N-terminus
- Signal recognition particle recognizes signal sequence while the protein is being translated.
- SRP-protein binds to SRP receptor on RER
- translation continues to the RER where the signal peptide is cleaved by signal peptidase
secretion
- some proteins enter secretory vesicles and are released from the cell or added to the membrane
lysosomes targeting
- mannose-6-phosphate -> clathrin coated vesicles -> endoscopes -> lysosomes
protein KDEL sequence targets where
- back to RER
hydrophobic proteins go where
- membrane
chaperones
- proteins that facilitate proper protein folding or prevent aggregation of newly-synthesized proteins
heat shock proteins
- induced by heat shock to help cellular protein refold properly and not become denatured
chaperonins
- CCT/Tric
- protein to be folded is enveloped by the CCT/Tric barrel and is folded using ATP
Diphtheria toxin
- B subunit facilitates entry of A subunit into the cell
- A subunit catalyzes the addition of ADP-ribose to EF2
- inhibits EF2 and protein synthesis
- leads to death
role of tetracycline
- binds to bacterial 30S subunit and blocks access of aminoacyl-tRNA to the A site
- reversible
role of puromycin
- resembles aminoacyl-tRNA
- accepts peptide chain and terminates translation
role of chloramphenicol
- binds to bacterial 50S subunit and inhibits peptidyltransferase
- can also inhibit human mitochondrial protein synthesis
role of erythromycin
- binds to the bacterial 50S subunit and inhibits translocation
role of streptomycin
- binds to the bacterial 30S subunit and prevents initiation
- also causes misreading of mRNA in which premature termination or incorporation of incorrect amino acids occurs.
what is nonsense mediated mRNA due to?
- mutations or mistakes in transcription or splicing where some mRNAs are defective and encode defective or truncated proteins
what happens in nonsense mediated mRNA decay?
- detects and degrades the aberrant mRNAs
what happens normally in mRNA?
- proteins normally bind the exon-intron junctions
- stop codon is in the last exon
- junctions are removed by initial ribosome movement and the mRNA exists the nucleus
- if all junctions are removed because the ribosome didn’t encounter a stop codon then the mRNA survives
what happens abnormally in nonsense mediated mRNA decay?
- premature stop codon encountered
- distal junctions remain
- mRNA degraded by Upf proteins
