RNA Flashcards
transcription
- RNA synthesized from the template DNA strand in a 5’ to 3’ direction.
- The coding strand is complementary to the template strand and shows what the RNA will look like, aside from having thymadine instead of uradine
messenger RNA
- longest chains of RNA
* nucleotides specify amino acids that are used to make proteins
ribosomal RNA
forms ribosomes (site of protein synthesis in cells)
transfer RNA
- transfers amino acids to proteins
* important for translation
micro RNA (miRNA)
- target mRNA molecules -> bind via base pairing -> remove poly-A tail -> mRNA degradation by endonucleases
- block translation into protein
small interfering RNA (siRNA)
- regulate gene expression
* cause degradation of mRNA
small nuclear RNA (snRNA)
splicing of pre-mRNA
RNA polymerase
- synthesizes RNA from DNA template by binding to promoter region and opening double helix
- does NOT require a primer
- requires transcription factors (proteins)
types of RNA polymerase
Eukaryotes:
•RNA polymerase I -> most rRNA (5.8S, 18S, and 28S)
•RNA polymerase II -> mRNA
•RNA polymerase III -> rRNA (5S) and other RNAs
Prokaryotes have only 1 RNA polymerase that is a multisubunit complex
alpha amanitin
- powerful inhibitor of RNA polymerase II
- from death cap mushroom (amanita phalloides)
- liver failure
Rifampin
- inhibits bacterial RNA polymerase
* used for tuberculosis
actinomycin D
•used in chemotherapy to inhibit RNA polymerase -> blunts replication of cancer cells
promoters
- DNA regions that are not transcribed
- bind to RNA polymerase and transcription factors
- binding to RNA polymerase opens double helix
common eukaryotic promoters
- TATA box (TATAAA, binds esp TFIID)
- CAAT box (CCAAT)
- GC box (GGGCGG)
Enhancers
- DNA sequences that increase rate of transcription
- can be upstream or downstream from gene
- bind to transcription factors called activators -> stabilize transcription factors/ RNA polymerase
- b/c DNA coiling, can be geometrically close to gene while many nucleotides away
Silencers
- DNA sequences that decrease rate of transcription
- can be up- or downstream of gene
- binds transcription factors called repressors -> prevent RNA polymerase binding
untranslated regions
- 5’ end -> upstream coding sequence and recognized by ribosomes to initiate translation
- 3’ end -> found after stop codon; importation for post-translational gene expression
significance of introns and exons
- Eukaryotic DNA has introns and exons that are transcribed into RNA within the nucleus
- before exiting nucleus the introns are cut out of the RNA
- only the exon portions enter cytoplasm to be translated to protein
- histone genes don’t have introns
heterogeneous nuclear RNA (hnRNA)
=pre-mRNA
•the initial transcript that is modified in nucleus to become mRNA
key modifications to mRNA before it leaves nucleus
- 5’ capping
- splicing out of introns
- 3’ polyadenylation
5’ capping
- addition of 7-methylguanosine to 5’ end soon after transcription begins
- distinguishes mRNA from other RNA
RNA splicing
- occurs during trancription
- removal of introns
- introns always have 2 nucleotides at either end: 5’ = GU and 3’ = AG
snRNPs
= small nuclear ribonucleaoproteins
•short RNA polymers with proteins
•RNAs have high content of uridine (U-RNA)
•5 U-RNAs: U1, U2, U4, U5, U6
spliceosome
= snRNPs + mRNA
•intron portion of mRNA forms loop called “lariat”
•lariat is released, then exons are joined
anti-SM (anti-smith)
- antibodies against proteins in snRNPs
* seen in lupus
anti-RNP
- antibodies against proteins in U1 RNA
- strongly assoc with Mixed Connective Tissue Disease
- also seen in lupus and scleroderma
alternative splicing
- allows many proteins to be made from the same gene by using a different combination of exons for translation
- allows eukaryotic cells to be more advanced and high functioning than prokaryotes
splicing errors
- loss of exons, retention of introns/incorrect joining of introns
- beta thalassemia - many mutations, but some involve splice sites
- oncogenesis - many splice site errors described
3’ polyadenylation
- triggered by polyadenylation signal (AAUAAA) which is followed by 10-30 nucleotides the CA
- once CSF and CstF bind, transcription is terminated
- then poly-A polymerase (PAP) binds and adds ~200 adenosines to the 3’ end (poly-a tail)
- NO template
Cleavage and polyadenylation specificity factor (CSF)
RNA binding protein that binds to AAUAA (polyadenylation signal)
Cleavage stimulation factor (CstF)
RNA binding protein that binds to CA sequence
poly-a polymerase (PAP)
the enzyme that binds after transcription has been terminated to add the poly A tails (~200 adenosine nucleotides) and removes part of the mRNA molecule
Processing bodies (P-bodies)
- organelles in cytoplasm
- some mRNA with less extensive miRNA binding will be sequestered here so its not translated
- mRNA often degraded, but some evidence shows it may be later translated
translation
- mRNA template -> protein
- occurs in cytoplasm on ribosomes
- tRNA brings amino acids to ribosome for protein assembly
ribosomes
- some free in cytoplasm, some part of rough ER
- contain rRNA and proteins
- large and small subunits
- size measured in svedberg units
svedberg units
- used to measure sized of ribosomes
* measure of the rate of sedimenation by centrifucation
prokaryotic ribosomes
•70S ribosomes •small units is 30S and large is 50S •small subunit: 16S RNA + proteins •large subunit: 5S RNA and 23S RNA + proteins (LOW yield)
protein synthesis inhibitor antibiotics
- target ribosomes of the size found only in bacteria
* ie aminoglycosides
Eukaryotic ribosomes
•80S ribosomes •small unit 40S and large unit 60S •small subunit: 18S RNA + proteins •large subunit: 5S RNA, 28 S RNA, 5.8S RNA + proteins (LOW yield)
tRNA
- transfer amino acids to protein chains
- synthesized by RNA polymerase III
- many bases are chemically modified
- cloverleaf shape (secondary structure) b/c base paring within molecule
- 70-90 nucleotides in length (tiny)
key portions of tRNA cloverleaf
- anticodon loop
- D loop (part of D arm)
- T loop (part of T arm
- 3’ end
anticodon of tRNA
- 3 nucleotides on tRNA
- pairs with complementary mRNA
- correct pairing -> correct protein synthesis
D loop of tRNA
- contains dihydrouridine
* recognized by aminoacyl-tRNA synthetase
T loop of tRNA
- contins T psi C sequence (TψC)
- T = ribothymidine
- psi = pseudouridine
- C = cytidine
- needed for tRNA ribosome binding
3’ end of tRNA
- always ends in CCA
* hydroxyl (OH) of A attaches to amino acid
charging of tRNA
- linking of amino acid to 3’ end of tRNA
- catalyzed by Aminoacyl-tRNA synthetase
- requires ATP
aminoacyl-tRNA synthetase in eukaryotes
in general there is a unique enzyme for every amino acid
hydrolytic editing
- aminoacyl-tRNA synthetase can also proofread the amino acid
- if incorrect it hydrolyzes it either from AMP or tRNA
protein synthesis direction
new amino acids are added the c-terminus of previos amino acids in protein synthesis
ribosome binding sites
- one for mRNA
- 3 for tRNA (they are codons in the mRNA)
- A-site: amino acid binding (anticodon) (3’)
- P-site: tRNA attached to growing protein chain
- E-site: exit of tRNA (5’)
initiation of translation
- begins with tRNA for methionine binding to the P-site at AUG start codon
- usually removed later by protease enzymes
- uses GTP hydrolysis
- in eukaryotes initiation factors are need to help assemble ribosomes and tRNA
N-formylmethionine (fMET)
- initiation codon AUG -> N-formylmethionine (fMET) in bacteria
- fMET in human bodies triggers chemotaxis of neutrophils (innate immunity)
elongation of translation
•uses elongation factors -> hydrolyze GTP to GDP
- charged tRNA binds to A-site
- amino acid joined to peptide chain (catalyzed by ribozyme activity called peptidyl transferase)
- ribosome translocation -> protein moved to p-site, a-site now empty, and tRNA in E site, ready to exit
- tRNA leaves E site
elongation factors in eukaryotes
- EF1 and EF2
* EF2 is the target of bacterial toxins -> inhibit protein synthesis (ie diptheria toxin and eotoxin A)
termination of translation
- ends at mRNA stop codon (UAA, UAG, and UGA)
- no tRNA anticodon for these codons -> no amino acid
- stop codon encountered -> releasing factors bind to ribosome -> catalyze addition of water to protein chain (add OH group)
posttranslational modification
- create functional protein
* includes folding and addition of other molecules
phosphorylation
- posttranslational modofication
* amino acid residue phosphorylated by protein kinase enzymes
glycosylation
- posttranslational modification
- formation of sugar-amino acid linkage
- N-, O-, C-linked glycosylation (sugar + nitrogen, oxygen, carbon)
- creates glycoproteins
hydroxylation
- posttranslational modification
- addition of hydroxyl (OH) groups
- important for collagen synthesis (hydroxilation of proline and lisine residues)
methylation of protein
- posttranslational modification
* addition of methyl (CH3) groups
acetylation of protein
- posttranslational modification
* addition of acetyl (CH3CO) group
ubiquitination of protein
- posttranslational modification
- addition of ubiquitin (small protein)
- tags proteins for destruction of proteasome
chaperones
- proteins that facilitate folding of other proteins
* classic example heat shock proteins
heat shock proteins
- aka stress proteins
- chaperones that are constitutively expressed, but levels increased with heat, pH shift, and hypoxia
- stabilized proteins/maintain structure
- helps cells survive environmental stress
mRNA start codon
AUG -> methionine in eukaryotes
school starts in AUGust
mRNA stop codons
UGA -> u go away
UAG -> u are going
UAA -> u are away
RNA stability
- stability/decay affects gene expression
- RNAs have varying half lives
- 3’ UTR is important in RNA stability/instability
Iron response elements (IRE)
- if low [iron] iron response proteins (IRPs) bind to IREs and repress ferritin translation while promoting transferrin receptor translation
- if high [iron] iron binds to IRPs and releases them from IREs -> ferritin translation occurs and transferrin receptor mRNA degraded
nonsense mediated decay (NMD)
•surveillance pathway to reduce errors in gene expression by eliminating mRNA
beta-thalassemia
- autosomal recessive inheritance
- anemia with reduced or absent synthesis of beta chains of hemoglobin
- depends on nonsense mediated decay pathway
Duchenne muscular distrophy (DMD)
- mutation on dystrophin gene
* nonsense mutation that causes mRNA decay and loss of functional protein
becker muscular dystrophy
•mutation on dystrophin gene, but does not involve a frameshift mutation
diamond blackfan anemia
- proapoptotic hematopoiesis, bone marrow failure, birth defects and predisposition to cancer
- cause by mutations in ribosomal proteins
treacher collins syndrome
- autosomal dominal craniofacial disorder
* mutation in TCO1 (treacle) -> role in ribosome biogenesis
small nucleolar RNAs (snoRNAs) mutations
mutations can cause prader-willi syndrome
chronic lymphocytic leukemia (CLL)
- most common leukemia in adults
* loss of part of chromosome 13 -> contains gene encoding miRNAs-15 and -16
