RNA Translation and Controlling Gene Expression

Question 1

tRNA: what is the anticodon?

Answer 1

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

Question 2

tRNA: where is the AA acceptor site?

Answer 2

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

Question 3

How is the tRNA for Valine named when it is unloaded and loaded with an AA

Answer 3

unloaded = tRNA_Val

loaded = Val-tRNA_Val

Question 4

What is the wobble base hypothesis

Answer 4

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

Question 5

What are the common wobble base pairings

Answer 5

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)

Question 6

In tRNA loading, how is an AA activated so it can be loaded onto a tRNA to create an aminoacyl tRNA?

Answer 6

AA + ATP → AA-AMP + PP_i

AA-AMP + tRNA → aminoacyl tRNA + AMP

Question 7

the _____ of AA and tRNA will power peptide bond formation

Answer 7

hydrolysis

Question 8

What are aminoacyl-tRNA synthetases?

Answer 8

enzymes that ensure the correct AA is paired w/ its correlating tRNA

Question 9

What is the prok. and euk. ribosome numbers?

Answer 9

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.

Question 10

Of the E, P, and A sites on the ribosome, what is the order in which tRNA molecules move through these sites?

Answer 10

A → P → E

Question 11

Does prokaryotic translation always have to occur at the 5’ site of the mRNA? Why or why not?

Answer 11

No, because prok mRNA is polycistronic

Question 12

What is the Shine-Dalgarno sequence?

Answer 12

the prok mRNA binding site where the ribosome attaches to start translation

Question 13

What are the basic steps in the initation of prok. translation?

Answer 13

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-tRNA_met

Question 14

Will all AUG on prok mRNA signify a start codon?

Answer 14

No. in order for AUG to be a start codon, it needs to be preceeded by a Shine Dalgarno sequence

Question 15

In the elongation step of prok. translation, _____ transferase catalyzes the formation between fMet & the 2nd AA

Answer 15

peptidyl transferase

Question 16

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?

Answer 16

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

Question 17

In the elongation step of prok. translation, what occurs during translocation?

Answer 17

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.

Question 18

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

Answer 18

1) A, A
2) peptidyl
3) ribosome

Question 19

It takes ___ high energy bonds to make a peptide chain

Answer 19

4n

n = # AA in the chain

Question 20

What sequence functions in starting euk translation?

Answer 20

5’UTR sequences (i.e. Kozak sequence)

these are usually situated a few nucleotides before the start codon

Question 21

Describe what happens during the initiation step in euk translation

Answer 21

Initation complex located the 5’ cap and scans for the start codon: 40S, Met-tRNA_Met, 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

Question 22

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

Answer 22

1) translation
2) post-translational

Question 23

Eukaryotes have 2 main elongation factor proteins: eEF-1 and eEF-2. What are the primary roles of each?

Answer 23

eEF-1: helps load aminoacyl-tRNA into the A site & catalyzes release of GDP

eEF-2: is a translocase

Question 24

In euk translation, what are the main roles of release factors eRF1 & eRF3 during termination?

Answer 24

eRF1: recognizes all 3 termination codons

eRF3 is a GTPase that helps eRF1 release the completed protein

Question 25

What is cap independent translation?

Answer 25

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

Question 26

For cap independent translation, the mRNA must have a(n) ______, a special nucleotide sequence? What does this sequence usually code for?

Answer 26

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

Question 27

_____ is the principal site of regulatin of gene expression in both euk and prok

Answer 27

transcription

Question 28

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.

Answer 28

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

Question 29

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

Answer 29

1) inhibited i.e. trp operon
2) stimulated i.e. lac operon

Question 30

What are the p and o regions of the lac operon?

Answer 30

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

Question 31

What are the Z, Y, and A genes of the lac operon?

Answer 31

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

Question 32

What are the crp and I gene in the lac operon?

Answer 32

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)

Question 33

adenylyl cyclase converts ATP to cAMP; cAMP binds to ____, which helps activate the RNA Pol. activity at the lac operon

Answer 33

CAP (catabolite activator protein), which is produced by the crp gene

Question 34

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

Answer 34

repressor protein, operator site

Question 35

How does increased glucose affect the lac operon?

Answer 35

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

Question 36

Trp operon: bacteria can make their own tryptophan via the trp operon. Is this transcription always on? Why or why not?

Answer 36

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.

Question 37

In the trp operon activated or inhibited in the presence of tryptophan?

Answer 37

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.

Question 38

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.

Answer 38

TATA, TATA

Question 39

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 ____

Answer 39

promoter

Question 40

Gene repressor proteins ____ transcription

Answer 40

inhibit

Question 41

RNA translocation: mRNA is not translated until it has reached its proper location in the cell. Why?

Answer 41

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.

Question 42

Control of gene expression at the RNA level in euk transcription: mRNA surveillance refers to ___ mRNA transcripts being degraded

Answer 42

defective

Question 43

RNA interference (RNAi): siRNA s bind complemetary sequences on mRNAs, & this ds-RNA is then ____.

Answer 43

degraded

Question 44

Post-translational modification of proteins: _____ assist in correct 3D folding of proteins

Answer 44

chaperones

Question 45

Post-translational modificaiton of proteins: name the various types of covalent modification that can be used to modify newly synthesized proteins

Answer 45

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 (PO₄^3-)

sulfation (SO₄^2-)