RNA Translation and Controlling Gene Expression Flashcards
tRNA: what is the anticodon?
a sequence of 3 nucleotides which is complementary to the mRNA codon the tRNA translates
the anticodon is responsible for recognizing the mRNA codon to be translated
tRNA: where is the AA acceptor site?
it’s at the 3’ end of the tRNA molecule and is opposite of the anticodon. This is where the AA attaches to the tRNA
How is the tRNA for Valine named when it is unloaded and loaded with an AA
unloaded = tRNAVal
loaded = Val-tRNAVal
What is the wobble base hypothesis
refers to that some codons pair with >1 anticodon/tRNA
the 1st 2 codon-anticodon pairs obey normal base pairing rulse, but the 3rd position is more flexible
What are the common wobble base pairings
G-U (and vice versa), I-U, I-A, and I-C
I = modified inosine base (inosine is derived from adenine)
Mneumonic: wobbly bases like GUAC (as in guacamole)
In tRNA loading, how is an AA activated so it can be loaded onto a tRNA to create an aminoacyl tRNA?
AA + ATP → AA-AMP + PPi
AA-AMP + tRNA → aminoacyl tRNA + AMP
the _____ of AA and tRNA will power peptide bond formation
hydrolysis
What are aminoacyl-tRNA synthetases?
enzymes that ensure the correct AA is paired w/ its correlating tRNA
What is the prok. and euk. ribosome numbers?
prok. = 70S (30S small subunit and 50S large subunit) with a 23S ribozyme
euk. = 80S (40S small subunit and 80S large subunit) with a 28S ribozyme
mneumonic:
prok. are odd little creatures. euk are even.
Of the E, P, and A sites on the ribosome, what is the order in which tRNA molecules move through these sites?
A → P → E
Does prokaryotic translation always have to occur at the 5’ site of the mRNA? Why or why not?
No, because prok mRNA is polycistronic
What is the Shine-Dalgarno sequence?
the prok mRNA binding site where the ribosome attaches to start translation
What are the basic steps in the initation of prok. translation?
1) 30S (small subunit) binds to proteins IF1 and IF3; this complex then binds to the mRNA transscript
2) the 1st aminioacyl-tRNA (aka initiator tRNA) and IF2 joins; IF2 is bound to GTP
3) 50S (large subunit) joins and completes the complex
- process is powered by 1 GTP
- initiator tRNA = met-tRNAmet
Will all AUG on prok mRNA signify a start codon?
No. in order for AUG to be a start codon, it needs to be preceeded by a Shine Dalgarno sequence
In the elongation step of prok. translation, _____ transferase catalyzes the formation between fMet & the 2nd AA
peptidyl transferase
In the elongation step of prok. translation, when peptidyl transferase catalyzes the formatio between fMet & the 2nd AA, what is considered the nucleophile, the electrophile, and the leaving group?
The carboxylic C on fMet is the electrophile and the N on the 2nd AA is the nucleophile (because the N attacks the C), while the tRNA is the leaving group
In the elongation step of prok. translation, what occurs during translocation?
tRNA in the P site moves to the E site, and the tRNA in the A site moves to the P site; then, the next codone followed by its anticodon moves into the A site. This process is repeated until a stop codon is reached.
Termination in prok. translation:
1) occurs when a stop codon appears in the ___ site of the ribosome, which signals a realse factor (not tRNA) to enter the ____ site.
2) _____ transferase hydrolyzses the bond between the last tRNA and the completed polypeptide.
3) the _____ separates and release both the mRNA and polypeptide
1) A, A
2) peptidyl
3) ribosome
It takes ___ high energy bonds to make a peptide chain
4n
n = # AA in the chain
What sequence functions in starting euk translation?
5’UTR sequences (i.e. Kozak sequence)
these are usually situated a few nucleotides before the start codon
Describe what happens during the initiation step in euk translation
Initation complex located the 5’ cap and scans for the start codon: 40S, Met-tRNAMet, and several eukaryotic initiation factors proteins (eIFs) binds to the 5’ cap; additional proteins are recruited, and then the whole complex scans for the start codon
1) the amount of eIF proteins present can either increase or decrease _____ rates
2) eIFs are controlled by _______ modification i.e. phosphorylation, which couples translation to upstream signaling pathways
1) translation
2) post-translational
Eukaryotes have 2 main elongation factor proteins: eEF-1 and eEF-2. What are the primary roles of each?
eEF-1: helps load aminoacyl-tRNA into the A site & catalyzes release of GDP
eEF-2: is a translocase
In euk translation, what are the main roles of release factors eRF1 & eRF3 during termination?
eRF1: recognizes all 3 termination codons
eRF3 is a GTPase that helps eRF1 release the completed protein
What is cap independent translation?
refers to the ability of eukaryotes to sometimes start translation in the middle of the mRNA molecule.
to accomplish this, the mRNA must have an internal ribosome entry site (IRES), a sepcial nucleotide sequence
For cap independent translation, the mRNA must have a(n) ______, a special nucleotide sequence? What does this sequence usually code for?
internal ribosome entry site (IRES)
most code for proteins that help the cell deal with stress or help activate apoptosis
activation of IRES requires different proteins than normal initiation
_____ is the principal site of regulatin of gene expression in both euk and prok
transcription
Controlling gene expression at the DNA level includes DNA methylation, chromatin remodeling, gene dose, imprinting, and X chromosome activation. Briefly describe what each of these are.
DNA methylation: in prok, methylation can either promote or inhibit transcription. in euk, methylation physically blocks the gene from transcriptional proteins and, therefore, turns off transcription
Chromatin remodeling: in euk, certain proteins bind methylated CpG groups and recruit chromatin remodeling proteins that change the winding of DNA around histones, which turns off transcription
Gene Dose: having a copy number variation via either gene amplification or gene deletion can increase or decrease quantities of protein made, respectively
Imprinting: genomic imprinting is when only 1 allele of a gene is expressed, which is an epigenetic process d/t silencing of a certain gene involving DNA methylation, histone modification, and binding of long ncRNAs. these epigenetic marks are established in the germline and maintained throughout life and mitotic division
X chromosome inactivation: for females, during early development at the blastocyst stage, each cell in the inner cell mass randomly inactivates an X chromosome (hence females only have 1 active X chromosome in every cell). this does not occur in males
1) repressible enzymes are anabolic enzymes whose transcription is ____ in the present of excess amounts of product i.e. ____ operon
2) inducible enyzmes are catabolic enzymes whose transcription is ____ by the abundance of a substrate i.e. ____ operon
1) inhibited i.e. trp operon
2) stimulated i.e. lac operon
What are the p and o regions of the lac operon?
p region: the promoter site on DNA to which RNA Pol. binds to initiate transcription
o region: the operator site to which the lac repressor binds to
What are the Z, Y, and A genes of the lac operon?
Z gene: codes for B-galactosidase (cleaves lactose into glucose and galactose)
Y gene: codes for permease (transports lactose into the cell)
A gene: codes for transacetylase (transfers an acetyl group from acetyl-CoA to B-galactosidase
What are the crp and I gene in the lac operon?
these are regulatory genes/sequences that code for protein products that control the gene expression of Z, Y, and A
crp gene: located at a distant site w/ its own promoter; codes for CAP (catabolite activator protein) & helps couple the lac operon to glucose levels in the cell
I gene: located at a distant site with it’s own promoter; codes for the lac repressor protein (which binds to the lac operator to prevent transcription)
adenylyl cyclase converts ATP to cAMP; cAMP binds to ____, which helps activate the RNA Pol. activity at the lac operon
CAP (catabolite activator protein), which is produced by the crp gene
lac operon: when there is presence of lactose in a cell, the lactose binds to the ____ protein, inducing a conformational change so that it can no longer sit on the ____ site. RNA Pol. can now transcribe the genes Z, Y, A
repressor protein, operator site
How does increased glucose affect the lac operon?
increased glucose decreases lac operon transcription: increased glucose inactivates adenylyl cylcase, which means there will be decreased cAMP levels, so little to no cAMP can bind to CAP to initiate RNA Pol to start transcription
Trp operon: bacteria can make their own tryptophan via the trp operon. Is this transcription always on? Why or why not?
No. If the bacteria are surrounded by an environment that contains tryptophan, then there is no need to waste energy making tryptophan, so the trp operon will be off.
In the trp operon activated or inhibited in the presence of tryptophan?
inhibited. when the environment contains excess tryptophan, the tryptophan will bind to the operon, which then prevents RNA pol. from initiating transcription of the 5 genes to make tryptophan.
Control of Gene Expression at the RNA level in eukaryotes: For protein coding genes, upstream elements (UCEs) contain a core promoter and a ___ box (highly conserved DNA recognition sequence) for the ____ box binding protein.
TATA, TATA
Control of Gene Expression at the RNA level in euk transcription: When the TATA box binding protein binds to the TATA sequence, it initiates the assembly of the transcription complex at the ____
promoter
Gene repressor proteins ____ transcription
inhibit
RNA translocation: mRNA is not translated until it has reached its proper location in the cell. Why?
this helps ensure the translated proteins are in the correct location of the cell, as cells are highly polarized (locations vary in charge), and can affect how proteins are located throughout the cell.
Control of gene expression at the RNA level in euk transcription: mRNA surveillance refers to ___ mRNA transcripts being degraded
defective
RNA interference (RNAi): siRNA s bind complemetary sequences on mRNAs, & this ds-RNA is then ____.
degraded
Post-translational modification of proteins: _____ assist in correct 3D folding of proteins
chaperones
Post-translational modificaiton of proteins: name the various types of covalent modification that can be used to modify newly synthesized proteins
acetylation (-C(O)CH3)
formylation (-C(O)H)
alkylation (i.e. methyl, ethyl group)
glycosylation (results in glycoprotein d/t addition of a saccharide)
phosphorylation (PO43-)
sulfation (SO42-)
