Chapter 5

Question 0

4 cellular roles for RNA

Answer 0

1. Intermediate bt the genes and the protein synthesis machinery (mRNA) 2. An adaptor bt amino acids and codons of mRNA (tRNA) 3. Structural role in ribosomes (rRNA)
2. Regulatory molecule during translation of mRNA
3. Enzymatic which catalyze reactions (ribozymes)

Question 1

Why does evolution choose uracil rather than thymidine in DNA?

Answer 1

C spontaneously become U which repair systems recognize as foreign to DNA and is restored to C

Question 2

1. Stem loop structures
2. Bulges
3. Internal loops
4. Junctions

Answer 2

1. Intervening RNA is looped out from the end of the double helical segment
2. An unpaired nucleotide in 1 side of the bulge
3. Unpaired nuceotides on either side of the stem
   4.

Question 3

A feature of RNA that adds to its propensity to form double helical structures is it’s ___ pairing. Why? What does this enhance RNA capacity to do?

Answer 3

Non Watson crick base pairing/noncanonical base pairing. Pairing bt GA (N3 of U bonded to c6 carbonyl on G and carboxyl on C2 of U and N1 of G) are most common. enhances RNA capacity of self complimentarily so there is local regions of base pairing but not long range double helical regularly, like DNA.

Question 4

RNA is prevented from forming a B form helix due to___. Rather, the double helical RNA resembles ___

Answer 4

2’ hydroxyls in the backbone, A form DNA with a wide shallow minor groove, making it accessible. However, minor grooves contain no info on A:U vs U:A, yet can differentiate bt A:U vs G:C. The major groove is so deep and narrow, that seqs aren’t accessible to any AA side chain of interacting proteins

Question 5

Because of RNA’s inaccessible major grooves and minor grooves that lack seq specificity, interacting protein rely on___ for recognition. (Examples)

Answer 5

Hairpin loops, bulges, distort ions caused by non-canonical bps.
Ex: tRNA synthetases w/ their trnas in ricin

Question 6

RNAs enormous amount of rotation freedom is due to:

Answer 6

Bc it has a backbone of non base paired regions

Question 7

Protein assist RNA in formation of tertiary structures by:

Answer 7

Shielding the negative charges of the backbone phosphates, whose electrostatic repulsion so would otherwise destabilize the structure/not allow its formation.

Question 8

RNA structure identification can be done via: __&__ (which cannot be used w large RNA molecules). They are traditional methods which aren’t very good anymore.

Answer 8

X-ray crystallography & nuclear magnetic resonance NMR

Question 9

Better choice for identifying RNA structure is to ___ the __ __ of an RNA molecule with chemicals that react with __ __ in combination algorithms that predict the structure from the known energies of __ and ___.

Answer 9

To PROBE the SECONDARY STRUCTURE of an RNA molecules with a chemical that reacts with the UNPAIRED BASES in combo with an algorithm that predicts structure based on the known energies of STACKING and HYDROGEN BOND INTERACTIONS.

Question 10

The best form of RNA structure identification is via mutate and map strategy. It’s a 2D procedure that combines __ & __ modification all approaches

Answer 10

Mutational and chemical

Question 11

Step for performing mutate and map strategy/SHAPE

Answer 11

1. Library of nucleotide substitutions is made in which each nuc is replaced with its counter nucleotide.
2. Each mutant RNA is chemically modified by a procedure known as SHAPE
   SHAPE:
3. RNA is treated w a chemical that preferentially acetylates the 2’OH of any unpaired nucleotides
4. The position of the unpaired nucs is determined using primer extension strategy where DNA primers are elongated using reverse transcriptase.
5. Reverse transcriptase ceases elongation when it encounters a chemical modification
6. The positions of the chemical modifications are then determined by the size of the primer extension products
7. Data analyzed using an RNA structure modeling algorithm.

Question 12

Bc of the SHAPE algorithm’s ability to predict RNA helical and long range interactions involving few adjacent nucs, the mutate and map strategy makes it possible to predict __ & ___ RNA structures.

Answer 12

Secondary and tertiary

Question 13

Ribose itches are regulatory RNA elements that:

Answer 13

Bind and respond to small molecule ligands in controlling gene transcription and translation

Question 14

Shared features of enzymes and ribozymes:

Answer 14

Each contains active site, binding site for a substrate, and a binding site for a cofactor (such as a metali on)

Question 15

What does RNase P do? How does it work? What is its composition? How do its moieties work?

Answer 15

It’s an endoribonuclease involving in generating tRNA molecules from larger precursor RNAs.

RNase P cleaves off leader segment from 5’ end of the precursor RNA to generate mature tRNA.

Composed of protein and RNA however the RNA moiety is what gives it the catalytic nature.

The protein moiety facilitates the reaction by shielding the neg charges on RNA so they can bind effectively to the neg charged substrate.
The RNA moiety is able to catalyze cleavage of the tRNA precursor in the absence of the protein only if a small positively charged counter ion (such as a spermidine peptide) is added to shield the charges.

Question 16

The site of cleavage of the phosphodiester bond backbone is located within ____, with the protein moiety interacting with___

Answer 16

The catalytic center of the RNase P RNA moiety, the leader.

Question 17

An ex of an RNA that is a ribozymes and a riboswitch is a structure found in the ____of mRNA for the protein enzyme ___, which catalyzes ___? How does it work?

Answer 17

5’-untranslated region, GlmS..the synthesis of the metabolite glucosamine-6-phosphate.

The RNA is a ribozymes that degrades the mRNA for GlmS. This ribozymes activity is dependent on glucosamine-6-phosphate levels. (When they are high, the mRNA is degraded, curtailing synthesis of the metabolite). Making it a ribozymes and a riboswitch.

Question 18

Many ribozymes perform ___ reactions which are involving in

Answer 18

Trans-esterification reactions, removing introns from precursors to certain mRNAs, tRNAs, and rRNAs in a process known as splicing.

Question 19

The hammerhead is a __ ribonuclease that is found in certain infectious RNA agents of plants known as__, which depend on self cleavage to propagate. How does the viroids’ mechanisms work?

Answer 19

Seq specific ribonuclease, viroids.

When a viroid replicates it makes multiple copies of itself in one single, continuous RNA chain. Single viroids arise by cleavage and ths cleavage rxn is performed by the RNA seq around the junction.

Question 20

The self cleaving hammerhead seq is named so because:

Answer 20

It’s secondary structure contains three base paired stems (I, II, III) surrounding a core of no complimentary nucs req for cleavage.

Question 21

How does the hammerhead work?

Can every ribose in the RNA chain do it?

Answer 21

Cleaves RNA via forming a 2’, 3’ cyclic phosphate. How? At high ph, the 2’ hydroxyl of ribose is deprotonated, yielding a neg oxygen atom. This O can attack the scissile phosphate of the 3’ position of the same ribose. This will break the RNA chain and produces a 2’, 3’ cyclic phosphate and a free 5’-hydroxyl. Each ribose in an RNA chain can perform this rxn, completely cleaving the parent molecule into individual nucs.

Question 22

Bc the normal function of the hammerhead is self cleavage, it is not really a __; each molecule usually promotes a reaction___. How can a hammerhead be engineered to act as a true ribozyme?

Answer 22

Catalyst, one time only thus having a turnover number = 1.

By dividing the molecule into two parts, one being the ribozyme that contains a catalytic core and the other being the substrate that contains the cleavage site. The substrate binds to the ribozyme at stems I and III and after cleavage the substrate is released and replaced by a fresh, uncut substrate…thereby allowing repeated rounds of cleavage.

Question 23

Most ribozymes act on ___ centers. However, peptidyl transferase, which is responsible for __, acts on a ___ center in catalyzing the rxn.

The fact that one of the most functional enzymatic rxns in a cell is catalyzes by RNA supports the hypothesis that:

Answer 23

Phosphorous center, peptide-bond formation during protein synthesis, carbon center

Contemporary, protein based life arose from an earlier RNA world.

Question 24

Scientists designed modified versions of the hammerhead as potential therapeutics. To target cleavage of another RNA molecule, describe how the engineered structure of the hammerhead is modified. Why does this change make it a true ribozyme?

Answer 24

Separated into two parts, one being capable of completing catalysis and the other being the substrate. Bc the substrate is not attached to the catalytic portion of the hammerhead, the substrate can be released to allow for a new molecule to bind in its place. The hammerhead is now a true ribozyme, being able to complete many rounds of the rxn.

Question 25

When a methyl group is added to the 2’hydroxl of the ribose in adenosine…which means that adenosine is not subject to____?

Answer 25

Oxygen can no longer become deprotonated at high pH and attack the scissile phosphate at the 3’ position of the ribose. This means that this nucleoside is not subject to hydrolysis.

Question 26

How does RNA double helical structure differs from DNA?

Why can’t RNA form B form helix like DNA can? Seq specificity?

Answer 26

May exhibit internal loops, bulges, junctions, non complimentary nucs. Also, the RNA helix may contain G:U pairing

The 2 OHs in the RNA backbone. Instead it resembles A form DNA. Thus, very little seq specific info given from RNA bc the major groove is so narrow and inaccessible.

Question 27

What properties of RNA would allow it to specifically bind to an antibiotic?

Answer 27

RNAs many secondary structures allow it to bind to small molecules and fluorophores

Question 28

You’re given a seq of RNA isolated as a viroid to a specific plant species, identify some characteristics that would indicate that this viroid acts as a catalytic hammerhead.

Answer 28

If the viroid has a magnesium ion near its catalytic center and is capable of performing repeated round of cleavage

Question 29

All nucleosides except adenosine are subject to_____in a strand of DNA bc a methyl group is added to the 2’ hydroxyl of ribose

Answer 29

Hydrolysis