Ch. 21

Question 1

What are the 4 bases in RNA?

Answer 1

• Adenine
• Uracil
• Cytosine
• Guanine

Question 2

Draw adenine.

Answer 2

Question 3

Draw guanine.

Answer 3

Question 4

Draw cytosine.

Answer 4

Question 5

Draw thymine.

Answer 5

Question 6

Draw uracil.

Answer 6

Question 7

Draw the modified nucleotides.

Answer 7

Question 8

Compare and contrast DNA and RNA.

Answer 8

DNA
- Thymine
- Deoxyribose sugar

RNA
- Uracil
- Ribose sugar
- Complex intrastrand structures
- Can form ribozymes (catalytic molecules)

Question 9

Why is RNA a highly dynamic molecule?

Answer 9

1. RNA undergoes a cycle of synthesis, functional interaction & degradation
2. Its structure is altered by binding of ligands
3. Base pairing between mRNA and ncRNA can modulate protein synthesis process

Question 10

What do the 3 types of RNA needed for protein synthesis do (mRNA, rRNA, tRNA)?

Answer 10

• mRNA: carrier of genetic info
• rRNA: major constituent of ribosomes
• tRNA: adaptor molecule that connects RNA synthesis to protein synthesis

Question 11

How are transcription and translation different in prokaryotes vs eukaryotes?

Answer 11

Prokaryotes
- Both transcription and translation occur in the same compartment –> mRNA is bound by ribosomes as soon as it’s transcribed
- Transcription and translation are tightly coupled

Eukaryotes
- Transcription occurs in nucleus, translation occurs in cytoplasm
- Requires additional factors to transport RNA from one compartment to the next

Question 12

What does monocistronic mean?

Answer 12

Encodes a single protein

Question 13

What does polycistronic mean?

Answer 13

Encodes multiple proteins on a single mRNA

Question 14

Is eukaryotic mRNA monocistronic or polycistronic?

Answer 14

Most often monocistronic
- Coding sequence is often discontinuous due to introns (don’t code for protein) interrupting exons (protein-coding)

Question 15

Explain eukaryotic mRNA processing.

Answer 15

• Introns must be removed from precursor mRNA via RNA splicing to generate an ORF
• 5’ terminus of mature mRNA m⁷G cap
• 3’ terminus contains poly A tail
• m⁷G cap and the poly A tail facilitate interaction between mRNA and ribosome, increasing translational efficiency

Question 16

What kinds of organisms have proportionally higher ncRNA?

Answer 16

Multicellular organisms (but inversely related to proportion of protein-coding genes)

Question 17

What are the 5 way lncRNA are located in the genome?

Answer 17

Question 18

What are the 4 modes of action lncRNA has in mediating cellular functions?

Answer 18

1. Base pairing between nucleotides in the lncRNA and the target RNA
2. Base pairing between lncRNA and single-stranded regions of DNA
3. Formation of functional ribonucleoprotein complexes similar to ribosomes and spliceosomes
4. Ligand-induced riboswitches that function in signaling pathways

Question 19

Compare and contrast the structure of prokaryotic and eukaryotic RNA polymerases.

Answer 19

• Both have the same 5 core subunits
• Prok: also has α₂ββ′ω structure
• Euk: another 7 auxiliary protein subunits

Question 20

What is the cAMP receptor protein (CRP)?

Answer 20

Bacterial transcription regulatory protein that upon binding cAMP binds to specific DNA sequences and stimulates transcription

Question 21

What are σ factors?

Answer 21

• Bind to DNA sequences known as -35 and -10 boxes
• Binds bacterial RNA polymerase and is required for the initiation of transcription

Question 22

How do σ factors work?

Answer 22

Targets RNA polymerase to the promoter by decreasing the affinity of the protein for nonspecific DNA sequences by a factor of 10⁴

Question 23

What are housekeeping genes?

Answer 23

Encode proteins and RNA involved in essential biochemical processes common to most all cells

Question 24

What is DNase I footprinting?

Answer 24

In vitro lab method for identifying DNA sequences that can function as binding site for sequence-specific DNA binding proteins

Question 25

Why are the -35 and -10 boxes called that?

Answer 25

Protected sequences were located ~10 and ~35 nucleotides upstream of the transcription start site

Question 26

What is the TATA binding protein (TBP)?

Answer 26

Binds to the sequence 5′-TATAAAA-3′ located ∼30 nucleotides upstream of the RNA polymerase II transcription start site

Question 27

What is the structure of the TATA binding protein?

Answer 27

- Single polypeptide chain that folds to form 2 symmetric halves - Halves flank DNA and generate a sharp bend as a result of 2 Phe residues on each side that insert into the minor groove and force a kink into the DNA backbone

Question 28

What does RNA synthesis use to generate an RNA trascript?

Answer 28

**DNA template strand** (DNA must be unwound by RNA polymerase in order to access the template strand)

Question 29

What are the steps of transcription?

Answer 29

1. RNA polymerase, directed by its bound σ factor, binds to the promoter - A closed complex and an open complex form in succession 2. Transcription is initiated within the complex, leading to a conformational change that converts the complex to the elongation form followed by movement of the transcription complex away from the promoter (promoter clearance) - The σ subunit dissociates stochastically (at random) as the polymerase enters the elongation phase

Question 30

What is a closed complex?

Answer 30

**Inactive** form of bacterial RNA polymerase holoenzyme (bound DNA is intact)

Question 31

What is an open complex?

Answer 31

**Active** form of bacterial RNA polymerase holoenzyme (bound DNA is intact but partially unwound near -10 sequence)

Question 32

What are the 3 main RNA processing steps from primary transcript to mRNA?

Answer 32

1. 5’ capping: by guanine-N7 methyltransferase 2. Splicing 3. Polyadenylation (in the end, the exons are joined to form a continuous sequence that specifies a functional polypeptide)

Question 33

What is the function of 5' capping?

Answer 33

- Protection against ribonucleases (less rapid degradation) i.e. prolongs mRNA lifetime - Aids in export from the nucleus and in translation

Question 34

What is splicing?

Answer 34

Multiple proteins derived from a single gene - Exons retained/skipped - Introns excised/retained - Exon-intron splice sites altered - Alterations in transcription start site or poly-A addition

Question 35

What are the effects of alternate splicing?

Answer 35

- Soluble vs membrane-bound proteins - ± phosphorylation sites - Subcellular targeting information - ± allosteric effector site - Affinity of a ligand for a receptor

Question 36

Explain polyadenylation.

Answer 36

- Most mRNAs have a poly-A tail that is 80-250 nucleotides long - Serves as a binding site for specific proteins - Confers a distinctive 3’ end structure - Poly-A tail & associated proteins likely help protect the mRNA from enzymatic degradation - Poly-A tail is added in a multistep process; mediates transcription termination - Transcript is extended beyond the site where the poly-A tail is to be added - Then it is cleaved at the poly-A addition site by an endonuclease at highly specific sites: 5’-AAUAAA-3’ which is about 30 nucleotides upstream of the cleavage site

Question 37

When are ribozymes regenerated?

Answer 37

After the trans cleavage of target RNA

Question 38

What do ribozymes do?

Answer 38

Mediate intramolecular or intermolecular cleavage reactions of RNA substrates

Question 39

What are the 2 primary mechanisms proposed for ribozyme-mediated phosphodiester cleavage?

Answer 39

1. Acid-base catalysis 2. Two-metal ion catalysis

Question 40

What is the hammerhead ribozyme?

Answer 40

Catalytic RNA that is capable of both cis and trans cleavage

Question 41

How are intronic or intergenic sequences from eukaryotic mRNA, rRNA, or tRNA removed?

Answer 41

By an RNA-catalyzed reaction using either: - A self-splicing cis reaction - A trans-splicing reaction mediated by spliceosomes

Question 42

What are spliceosomes?

Answer 42

Ribonucleoprotein complex that **excises introns from mRNA and splices together the exons** to form a mature mRNA

Question 43

What are group I introns?

Answer 43

- Class of catalytic RNAs that use a **guanosine cofactor to self-splice** the introns and join the exons to form mature RNA - Found in precursors to rRNA, tRNA, and mRNA - Not in vertebrates

Question 44

What are group II introns?

Answer 44

Class of catalytic RNAs that don't need a guanosine cofactor for self-splicing, and instead **use a cis-acting adenine residue** within the intron

Question 45

What are 2 notable differences between group I and group 2 introns?

Answer 45

1. Group II introns do not require an **exogenous nucleoside** to initiate the cleavage reaction 2. Group II excised intron forms a **lariat** structure in which the 5′ end of the intron is linked to the 2′-OH of an adenosine residue near the 3′ end of the intron

Question 46

Diagram the mechanism of spliceosome-mediated trans splicing of precursor mRNA transcripts.

Answer 46

Question 47

What do introns removed by spliceosomes from primary mRNA transcripts contain?

Answer 47

Short conserved sequences at the 5′ (GU) and 3′ (AG) splice sites, as well as in the branch site

Question 48

What are small nuclear ribonucleoproteins (snRNPs)?

Answer 48

A complex of small nuclear RNAs and associated proteins that compose a spliceosome

Question 49

Diagram and explain the spliceosome assembly pathway.

Answer 49

1. Capped pre-snRNA transcripts are exported from the nucleus to the cytoplasm 2. Each snRNA is assembled with a heptameric ring of proteins, called Sm proteins, to form the snRNP core complex 3. snRNA is hypermethylated on the 5' cap and transported back to the nucleus 4. snRNP is assembled into a spliceosome complex

Question 50

What are common modifications in tRNA?

Answer 50

Question 51

What is small nucleolar RNA (snoRNA)?

Answer 51

Small RNA molecules that form part of the ribonucleoprotein complexes in the nucleolus that aid in processing rRNA molecules

Question 52

What are the 2 classes of alternative splicing mutations?

Answer 52

- Those that **disrupt the use of alternative splice sites (cis effects)** - Those that **affect expression of spliceosome components or regulatory proteins (trans effects)**

Question 53

What is the most frequent effect of alternative splicing mutations?

Answer 53

**Exon skipping** and occurs as a result of a base change at one of the conserved regions that must be recognized by the spliceosome complex

Question 54

What is retinitis pigmentosa?

Answer 54

Disease of the retina resulting in progressive retinal degeneration, which in some cases is caused by mutations in genes that encode spliceosome components

Question 55

Diagram mRNA decay.

Answer 55