Chapter 23- RNA Synthesis-DNA Transcription Flashcards
mRNA, tRNA, and rRNA are functionally different but share what and transcribed from what?
chemically identical but functionally different molecules of RNA transcribed from DNA anti-sense strand
prokaryotic ribosome have what s units?
5s, 16s, 23s rRNA
eukaryotic ribosome have what s units?
5s, 5.8s, 18s, and 28s
ribosomal RNA have sequence complementarity to regions of
mRNA
rRNA is a structural component of
ribosome
tRNA bind what to its ends?
mRNA to 5’ and amino acid to 3’
tRNA acts as an adapter to carry amino acid elements of protein as coded for by what?
mRNA
The 5’ terminus of tRNA is what?
base paired
The 3 ‘ terminus of tRNA is what?
a four base single stranded region, XCCA-3’-OH= CCA (acceptor stem)
Where is the site of amino acid attachment on the tRNA?
3’ CCA on the adenine
tRNA is made up of what modified bases?
dihydrouridine (DHU), ribosylthymine (rT), pseudouridine (Ψ), Inosine (I)
most abundant RNA?
rRNA (80%)
least abundant RNA?
mRNA (5%)
what does mRNA do?
carry the information stored in DNA in the nucleus to the cytoplasm where ribosome can make it into protein
What codon initiates translation?
AUG (methionine)
What codons terminate translation?
UAG, UGA, UAA
what is polycistronic mRNA and who has this?
polycistronic (polygenic), carry information for synthesis of several peptides from a single mRNA; only prokaryotes have this
mRNA in eukaryotes contain?
information for only a single polypeptide, contain exons (after introns spliced out)
what is pre-mRNA?
hnRNA (heterogeneous RNA)
where does translation occur in eukaryotes?
to cytoplasm on ribosomes
What does post translational modification include?
addition of 7-methylguanosine cap, poly A tail (AAAAA), and splicing (alternative splicing, RNA editing)
small nuclear RNA (snRNA) mediate what?
processing of large precursor molecules (primary transcripts) of mRNA, tRNA, and rRNA within the nucleus to produce the functional molecules for export to the cytosol
RNA synthesis is very similar to DNA synthesis except that
RNA synthesis does not require a primer to initiate transcription and 5’-end of growing RNA molecule has a triphosphate (for cap)
What are distinct features of eukaryotic transcription?
- Transcription initiates from many more sites than replication
- There are many more molecules of RNA polymerase per cell than DNA polymerase
- RNA polymerase proceeds at a rate much slower than DNA polymerase (approx. 50~100 bases/sec for RNA vs. near 1000 bases/sec for DNA)
- The fidelity of RNA polymerization is much lower than DNA. This is allowable since the aberrant RNA molecules can simply be turned over and new correct molecules made
Describe features of e.coli RNA polymerase
consists of 5 subunits (α2ββ’σ-holoenzyme)
Describe the subunits of e.coli RNA polymerase
β’-DNA binding
β-binds rNTP substrates and interact with σ
σ-recognize promoter sequences on DNA
w/o σ holoenzyme is called core enzyme
Characteristics specific to prokaryotic transcription initiation
no primer is required
after initiation, RNA polymerase holoenzyme loses the σ subunit and core enzyme (α2ββ’ subunits) carries out elongation
Characteristic of transcription elongation
mRNA is synthesized 5’ to 3’ direction from template strand (mRNA sequence is homologous to sense DNA strand)
Name 4 inhibitors of transcription initiation
Rifamycin B, Rifampin (rifampicin), Actinomycin D, and amanitin (toxic product of Amanita phalloides mushrooms
MOA of Rifamycin B
binds to β subunit of RNA polymerase, block binding of incoming rNTP at the initiation site preventing transcription initiation
MOA of Rifampin (rifampicin)
prevents the translocation of RNA polymerase along the DNA template thereby inhibiting transcription initiation
Can inhibit eukaryotic mitochondrial RNA polymerases but requires higher concentrations
Does rifamycin B and rifampin inhibit eukaryotic RNA? What can these be used to treat?
No, Since these compounds do not inhibit eukaryotic RNA polymerases, they have proven useful in the treatment of tuberculosis and gram-positive bacterial infections
MOA of Actinomycin D and what is it used to treat?
an intercalater that inhibits transcription initiation by binding to DNA, useful drug for treating childhood neoplasms (Wilms’ tumor) and choriocarcinoma
Termination sites of RNA synthesis have what characteristics?
- An inverted repeat base sequence containing a central non-repeating segment
- High G+C content sequence
- High A+T content sequence that result in 6~8 Uracil in RNA
What are subregions of prokaryotic promoter sites in RNA synthesis?
-35 sequence
Prinbow box
Two kinds of events in prokaryotic transcription termination
- self-terminating (dependent on the DNA base sequence only, termination factor independent)
- those that require termination protein Rho (termination factor dependent)
What is the action/function of Rho protein?
Rho binds to DNA:RNA hybrid region of a transcription bubble -> through its helices activity unwinds the newly synthesized RNA from the bubble -> terminates transcription
What happens in Rho independent termination?
inverted repeat DNA sequences are copied to RNA sequences -> then forms hair pin loop-> causes RNA polymerase to stop polymerization and terminal the transcription process
What happens if you eat mushrooms containing Amanitin?
amanitin a toxic cyclic octapeptide lead to rapid inhibition of RNA polymerase II and III and manifest mainly in liver. After 6-24 hours violent cramping and diarrhea set in, by 3rd day false remission sets in. On 4th or 5th day death will occur unless a liver transplant is done
Describe the 4 characteristics specific to eukaryotic RNA transcription
- Transcription and Translation in Eukaryotes are separated in space and time: enables eukaryotes to regulate gene expression in much more intricate ways, contributing to the variety of eukaryotic forms and functions
- RNA is stabilized through post-transcriptional modification steps: 5’ capping, splicing, poly A tailing
- RNA is synthesized by three types of RNA polymerases: RNA Pol I, II, III
- Promoters contain a TATA box near the start site for transcription
What are the 3 classes of eukaryotic RNA polymerases and what are they for?
- Pol I- for rRNA (nucleolus)
- Pol II- for mRNA and snRNA
- Pol III- for tRNA, 5s rRNA
What are the sensitivities of eukaryotic RNA polymerases to amanitin?
Pol I- amanitin insensitive
Pol II- strongly inhibited by amanitin
Pol III- inhibited by high amounts of amanitin
Are any of the eukaryotic RNA polymerases sensitive to rifampin?
eukaryotic pol is typically insensitive to Rifampin but “mitochondrial RNA pol” is sensitive
Describe eukaryotic promoter sites
Have complex promoters; TATA box and CAAT box
Many non-pol factors (transcription factors) required for binding of the enzyme to DNA
Describe eukaryotic holoenzymes
Huge holoenzymes; 2 large subunits, <10 small subunits
What structural features do RNA pol I, II, and III have in common?
- All 3 are big, multimeric proteins (500-700 kD)
- All have 2 large subunits with sequences similar to β and β’ in E. coli RNAP, so catalytic site may be conserved
- All have subunit homologs of α and ω in E. coli RNAP
These features are shared by RNAPs across species
What is the general function of TFIID
Binds TATA box, bends DNA
Regulatory targets, one binds Inr (initiator element)
What is the general function of TFIIA
Stabilizes (TBP-TATA complex) promoter binding by TBP (TATA binding protein)
What is the general function of TFIIB
Recruits RNAPII, defines start site
What is the general function of TFIIF
Binds RNAPII; destabilizes non-specific DNA interactions of RNAPII, positions template during initiation
What is the general function of TFIIE
Assists recruitment and activity of TFIIH, promoter melting
What is the general function of TFIIH
ATPase, CTD kinase, helicase
Promoter melting and escape
What factors does RNA pol II require to bind?
Gene specific transcription factors (TFIID, TFIIA, TFIIB, TFIIF, TFIIE, TFIIH)
TFIID binds to TATA binding protein (TBP) and associates with what?
TBP associated factors (TAFs) that mediate interactions with TBP
After binding TFIID to TBP what happens
TATA-TBP complex bends DNA-> TFIIA binds and stabilizes complex -> TFIIB binds to the DNA downstream of TATA-box-> stabilizes TBP-TATA complex-> formation of DAB complex
How is TFIIB recruited?
TFIIB binds to the DNA downstream of TATA-box
TFIIB makes specific contacts with the edge of the TBP saddle opposite that contacted by TFIIA, and makes non-specific interactions with the phosphate backbone of the DNA.
After the formation of the DAB complex what are the next steps to elongation?
formation of DAB complex -> binding of RNA polII-IIF complex -> binding of IIE and IIH -> elongation (a factor IIS is seen to stimulate the elongation reaction, it may be IIF also has a role in elongation)
What are components of eukaryotic mRNA processing?
- 5’ caps prevent 5’-3’ exonuclease 7-methyl G joined to 5’ of RNA
- 3’ tails
Cleavage of 3’ end signaled by poly A addition sequence (AAUAAA)
Poly A polymerase adds AMP (Non-template directed)
Tails bound & protected by cytoplasmic poly A binding protein (PABP)
Describe steps in splicing
Introns are spliced out sequentially from 5’ to 3’
1. Boundary sequences bind snRNPs
2. 5’ G trans-esterified to give lariat with 2’-5’ linkage and
3’ OH of exon 1
3. 3’ OH of exon 1 displaces intron 3’ end releasing intron lariat
This whole process is orchestrated by splicosomes which is very complicated
Describe alternative splicing
By altering how the pre-mRNA is spliced, different versions of the mRNA and ultimately, different proteins are synthesized
What is an example of eukaryotic protein that uses alternative splicing?
immunoglobulin synthesis
Describe self splicing introns
There are two types;
- Group I (in Tetrahymena (a ciliated protozoan))
- Group II (in mitochondrial and chloroplast genes)
What is an example of RNA editing?
Apo B gene codon # 2153 (CAA) transcription:
in liver produces Apo B-100 (no editing)
In intestine CAA codon edited via cytidine deaminase -> converts CAA (glutamine) to UAA (stop) -> forming Apo B-48
Termination of transcription in eukaryotes occurs how?
It is not known if termination factors are involved like prokaryotes.
Transcription does not stop at the poly A site but continues for another 200-2000 bases beyond the site before it is aborted
Proposed model:
nascent RNA is cleaved at an upstream part of the ‘termination region’ -> RNA polymerase is released from the DNA
What is the genetic code?
“Universal” genetic code consists of 64 triplets of bases, sets of three bases (triplets) on mRNA is a codon
each codon specifies and amino acid, or “start” or “stop”
What is the genetic code used for?
protein synthesis; translation of base sequence into amino acid sequence
What is different between standard tRNA and mitochondrial tRNA?
structure is different
Because most of the amino acids represent more than one codon, results in
61 codons correspond to 20 different amino acids
What are the functions of codon AUG
2 functions:
- code for Met
- signal to initiate protein synthesis
GUG has same functions, but rarely used
What are the codons that signal to terminate the growing polypeptide chain?
UAA, UAG, UGA
Most amino acids are represented by more than 1 codon except for
Met (AUG) and Trp (UGG)
How is fidelity of RNA translation determined?
Attachment of the correct amino acid to a particular tRNA molecule by the corresponding aminoacyl synthetase: the specificity of interaction of the enzyme, amino acid, tRNA
Correct codon-anticodon pairing: base pairing provide specificity
What is the wobble hypothesis?
Because there are 64 codons specifies amino acids but only 45 tRNAs the Anticodons of some tRNAs can recognize two or more codons:
U in anticodon can base pair with either A or G in codon
I (Inosine) in anticodon can base pair with A, C, or U
Affects base pairing of the 3rd base of codon, only
1st and 2nd base of codon follow Watson-Crick base pairing A=U, G=C
Application of wobble hypothesis using ACI codon
AC will follow watson crick pairing and I can pair with A, U, or C, therefore tRNA anticodon can recognize
UGA codon
UGU codon
UGC codon
Class I aminoacyl-tRNA synthetase include what a.a.
simple amino acids (e.g. Arg, Cys, Glu, and Ileu) added to 2’OH of ribose sugar which is ultimately on 3’ end of tRNA
Class I aminoacyl-tRNA synthetase include what a.a.
more complex amin acids (e.g. His, Phe, Pro) added to 3’OH of ribose sugar which is ultimately on 3’ end of tRNA
Two important functions of aminoacyl-tRNA?
- Absolute specificity in coupling amino acid to its cognate tRNA
- Activation of amino acid so that it readily reacts to form a peptide bond
Two step reaction of aminoacyl-synthetase is
the first, an activation step produced enzyme-bound aminoacyl-adenylate is highly, but not absolutely specific.
The second reaction which forms the aminoacyl-tRNA is absolutely specific (if incorrect amino acid is attached, tRNA is passive and will add that a.a. to growing polypeptide chain)
What are features that determine specificity of tRNA?
- A base in the anticodon
- One or more of three base pairs in the acceptor stem
- Base at position 73 (3’end of tRNA) preceding the CCA end
Ribosomes are present where?
cytosol or RER
Ribosomes composed of
rRNA and protein two subunits (Large/Small)
Prokaryotic ribosomes are
50s large, 30s small and 70s complete
Eukaryotic ribosomes are
60s large, 40s small, and 80s complete
Ribosomes serve as
workbench of protein synthesis, brings mRNA and tRNA together
Initiation of prokaryotic protein synthesis begins by
association of mRNA, formulated tRNA and ribosomes, and mediated by initiation factors (IF)
What are prokaryotic initiation factors?
IF-1, IF-2, IF-3
What is the function of IF-1?
cause dissociation of 70S ribosomes into 30S+50S
What is the function of IF-2?
carries out its function in forming the 30S initiation complex by having a GTP bound to it
What is the function of IF-3?
keeps the two forms (30s and 50s) from reassociating until the time is correct
What is the [shine-delgarno] sequence?
precise placement of the 30S subunit at the start codon is achieved by a base pairing between the 3’ end of the 16S rRNA of the 30S subunit at [sequences found just upstream from the initial AUG]
Describe the 70s initiation complex
binding of 50s to 30s complex, with loss of IF-1 and IF-3
Dissociation of IF-2 with hydrolysis of GTP to GDP and Pi
then tRNAmet occupies P site
Describe eukaryotic protein synthesis
Require 5’methylated cap
40S subunit start at the 5’end and “scans” down the mRNA until it detects the start codon-AUG
What is the eukaryotic consensus sequence for protein initiation?
CCRCCAUGG, where R is a purine (A or G)
What is required for the formation of the initiation complex in eukaryotic protein synthesis?
40S subunit, GTP, eukaryotic initiation factors (eIFs), and initiator tRNA are required
Mechanism of the formation of the initiation complex in eukaryotic protein synthesis
40S + eIF-3 to form 40SN structure -> join with the initiator tRNA -> Along with eIF-2, tRNA joins to form the 43S complex -> then binds to capped mRNA
Under the direction of eIF-4, 43S complex scans down the mRNA until it reaches start codon, the 48S complex
60S subunit joins by the action of eIF-5 to form the final 80S initiation complex
How does 40s recognize the 5’ cap?
eIF4F recognizes 5’ cap and consists of eIF-4E (cap binding protein), eIF-4A and eIF-4G
How does polio effectively translate its RNA if polio virus is not capped and methylated?
destroys the eIF-4G (stabilizing protein of cap binding complex eIF-4F), then uses its viral RNA internal ribosome entry site (IRES)
therefore disables host translation but activates translation of polios own translation
entry of next tRNA to A-site is carried out by
Carried out by EF-Tu (EF in eukaryotes)/GTP
Recycling of EF-Tu by EF-Ts (EF1b in eukaryotes)/GTP
Steps in elongation
entry of the next tRNA to the A-site -> formation of the peptide bond -> translocation -> termination
How is the formation of the peptide bond in translation done?
Peptide bond formation between the COOH of a.a. or peptide on the P-site tRNA and the NH2 of A-site tRNA
Catalyzed by a part of large subunit of the ribosome
How is translocation achieved in translation?
Move down the mRNA in the 5’ to 3’ direction
Requires another factor, EF-G (EF2 in eukaryotes)
Bring the next tRNA into the A-site using EF-Tu/GTP
How is termination achieved in translation?
Ribosome reaches one of the three stop codons: UAG, UAA, or UGA
Stop codons were identified by mutations in bacteriophage T4
Termination event is the same in both eukaryotes and prokaryotes except for the number of factors required: RF vs. RF1,2,3 only
How is new polypeptide chain brought to ER?
via signal recognition particle
transmembrane proteins can be synthesized how
SRP bound to ribosome and polypeptide sequence -> brings it to the membrane -> translates protein -> hydrophobic/hydrophilic properties -> create transmembrane protein (single pass, or multiple pass)
What is the role of GTP in protein synthesis?
GTP facilitates binding of protein factors either to tRNA (IF-2) or ribosomes (EF-Tu)
Hydrolysis of GTP to GDP in protein synthesis causes what?
dissociation of the bound factor
When GTP bound to factors induces what?
conformational changes
When is GTP active and inactive in protein synthesis?
GTP bound: active conformation
GTP hydrolyzed: inactive form
Molecular switch: GTP-on, GDP-off (ex. Cdc42)
What is Mucolipidosis II or I-cell disease
mislocalization of lysosomal enzymes (acid hydrolases)-lacks mannose-6-phosphate residues, cause indiscriminate destruction of tissues
α1-antitrypsin deficiency
defects in the secretion of α1-AT, a serine protease inhibitor-leukocyte protease (elastase), which may be released during inflammatory reactions in the lung leading to chronic lung (emphysema) & liver (cirrhosis) damage because unable to inhibit proteases
How do you identify the genetic variant of α1-antitrypsin
isoelectric focusing (gel electrophoresis)
MOA of Aminoglycosides:
interfere with binding to formyl-tRNA to ribosome, thereby preventing correct initiation of protein synthesis
irreversibly bind to 30S subunit (protein P10) to cause 1. Depletion of the ribosomal pool, 2. Formation of “ streptomycin monosomes”, 3. Misreading of mRNA leading to faulty proteins, which accumulate in the membrane and cause membrane damage/leak
MOA of Puromycin:
causes premature termination of chain, resembles tyrosinyl-tRNA
MOA of Tetracyclines:
reversibly inhibit AA-tRNA binding to the 30S subunit
MOA of Macrolides:
bind to 50S subunit near P-site to cause conformational change, no peptide bond formation, inhibit translocation (A-site to P-site)
MOA of Lincosamides:
bind near P-site and interfere with binding of aminoacyl end of AA-tRNA, occupy the site or destabilize ribosomes so growing chain fall off the mRNA
MOA of Chloramphenicol:
bind reversibly to 50S subunit near P-site and interfere with binding of aminoacyl end of AA-tRNA, inhibits peptide bond formation between AA-tRNA and growing peptide chain on P-site by inhibiting peptide transferases
Examples of aminoglycosides
Streptomycin, Neomycin, Kanamycin, gentamicin
Action of abrin, ricin on eukaryotes
inhibits binding of aminoacyl tRNA
Action of anisomycin on eukaryotes
inhibits peptide transferase on the 80s ribosome
Action of diphtheria toxin on eukaryotes
catalyzes a reaction between NAD+ and EF-2 to yield an inactive factor; inhibits translocation
Action of chloramphenicol on eukaryotes
inhibits peptide transferases of mitochondrial ribosomes; is inactive against cytoplasmic ribosomes
Action of puromycin on eukaryotes
causes premature chain termination by acting as an analogue of charged tRNA
Action of fusidic acid on eukaryotes
inhibits translocation by altering an elongation factor
Action of cycloheximide on eukaryotes
inhibits peptidyltransferases
Action of pactamycin on eukaryotes
inhibits positioning of tRNAfMet on the 40s ribosome
Action of showdomycin on eukaryotes
Inhibits formation of the eIF2-tRNAfMet-GTP complex
Action of sparsomycin on eukaryotes
inhibits translocation
Diphteria Toxin:
single polypeptide chain -> binds to outer surface of cells & enters by receptor mediated endocytosis, then cleaved into fragments -> responsible for inactivation of EF-2 by ADP ribosylation, specific to eukaryotic EF-2
Cholera Toxin:
Gsalpha- activate adenylate cyclase by ADP-ribosylation, activated enzyme catalyze cAMP formation from ATP, cAMP stimulate secretion of water and electrolytes from intestinal epithelial cells
Lectins:
a group of proteins that bind carbohydrates and agglutinate animal cells such as abrin, ricin, modeccin, highly toxic to eukaryotic cells
Inhibits protein synthesis by irreversibly inactivating EF-2 binding region of 60S ribosome subunit
each polypeptide chain of collagen is what helix
left handed helix, 3 a.a. per turn, 3 polypeptides form a right handed triple stranded super helix
What are the two stages in collagen biosynthesis and explain each
Intracellular: synthesis and assembly of the procollagen molecule
Extracellular: transformation of procollagen to collagen & incorporation into a stable cross-linked structure (Fibril)
