Biochem 432: RNA

Question 1

Describe the 5’ and 3’ ends of mRNA in eukaryotes

Answer 1

The 5’ end is capped with 7-methylguanylate

The 3’ end is extended by a poly A tail

Question 2

What is meant when it is said that Eukaryotic mRNA has a discontinuous coding sequence?

Answer 2

There are multiple reading frames within one stretch of mRNA.

Introns are sequences that interrupt RNA that must be removed from the precursor mRNA

Exons are protein coding sequences

Question 3

What are the steps of the 5’ capping mechanism for mRNA?

Answer 3

1. RNA polymerase II transcripts begin with 5’-triphosphate on the first nucleotide
2. Triphosphatase removes the terminal phosphate on the 5’ nucleotide
3. RNA Guanylytransferase transfers GMP from a GTP to the diphosphate end of the pre-mRNA
4. Guanine-N7 methyltransferase transfers a methyl group to the gap guanine to form a 7-methylguanosine with a 5’,5’ triphosphate linkage to the terminal nucleoside

Question 3

What are the four classes of introns?

Answer 3

Group I & Group II introns (self-splicing)

Spliceosomal introns (require splicesomes) (most common)

tRNA introns

Question 4

Compare and Contrast Group I & Group II introns

Answer 4

Group I introns are initiated by the binding of an essential exogenous guanosine cofactor whereas Group II introns do not require an exogenous nucleoside to initiate cleavage reaction—-instead there is an adenosine at the branch site.

Group II introns form a lariat (5’ end of the introns are linked to the 2’-OH of an adenosine) structure whereas group I do not because the 3’-OH of guanosine initiates a nucleophilic attack on the phosphodiester bond at the 5’ splice site to begin the first transesterification reaction.

Question 5

Describe a Spliceosome

Answer 5

Spliceosomes are ribonucleoprotein complexes. Eukaryotic spliceosomes are composed of small nuclear RNAs (snRNAs) and proteins, which together are called small nuclear ribonucleoproteins (snRNPs).

The snRNPs are named after the snRNA molecules contained within each RNA–protein complex

RNA in snRNP is called snRNA (for small nuclear RNA), which are 70−200 nt long. There are five snRNAs known in eukaryotes (U1, U2, U4, U5, U6).

Question 6

What gives “signal” for the removal of introns by spliceosomes

Answer 6

Introns removed by spliceosomes from primary mRNA transcripts contain short conserved sequences at the 5′ (GU) and 3′ (AG) splice sites, as well as in the branch site. These are similar to the sequences observed in the group II intron.

The branch site in the introns of primary mRNA transcripts is located 18–40 nucleotides upstream of the 3 for the formation of a lariat structure

Question 7

Explain the steps of Spliceosome-mediated precursor mRNA splicing

Answer 7

1. Spliceosome-mediated splicing consists of two transesterification steps, which releases the excised lariat intron and the spliced exonic sequences.
2. The invariant G from the 5′ end of the intron is linked to the branch point A through a 2′,5′ phosphodiester bond, forming a lariat structure.

Question 8

Explain the steps of the spliceosome reaction cycle

Answer 8

Step 1: the **recruiting ** of U1 and U2 snRNPs to the 5’ splice site;

Step2: **U2 associates ** with the branch site, forming the pre-spliceosome complex. The interaction is mediated by the snRNAs in both U1 and U2 snRNPs.

Step 3: U4-U5-U6 form a tri-snRNP complex, which is then recruited to the transcript to form the precatalytic spliceosome complex;

Step 4: Binding of the tri-snRNP destabilizes the U1 and U4 snRNPs, releasing them from the spliceosome;

Steps 5 and 6: the transesterification reaction;

Step7: mRNA release;

Step8: the U2-U5-U6 complex dissociates from the lariat intron to initiate another round of splicing.

Question 9

Explain/Describe polyadenylation of eukaryotic mRNA

Answer 9

— The precursor mRNA sequence that promotes polyadenylation is 5’-AAUAAA-3’, bound by the cleavage and polyadenylation specificity factor (CPSF);

— The downstream G and U-rich region is bound by the cleavage stimulatory factor (CStF);

— Binding of poly(A) polymerase, which synthesizes the poly adenine tail.

Question 10

What are the purposes of the 5’ cap and 3’ poly A?

Answer 10

Both stabilize and protect mRNAs

Aid in the export from the nucleus

Facilitated the interaction between mRNA and the ribosomes, increasing translation efficiency

They are used during translation to make sure the mRNA is intact

5’ cap and 3’ poly A occur in the nucleus. Addition of the cap occurs soon after initiation.

Question 11

Describe the changes that occur in the processing of tyrosine pre-tRNA

Answer 11

—pre-tRNAs undergo cleavage and extensive base modification

Cleavages:
—16-nucleotide 5’ end sequence cleaved by RNase P
—14-nucleotide intron in the anticodon loop removed by splicing

Base modications (10% modified)
—3’ end U residues replaced with a CCA sequence which is required for tRNA charging with amino acid by aminoacyl-tRNA synthetase
—Methyl and isopentenyl groups are added to specific residue purine base heterocyclic rings and ribose 2’-OH groups
—Specific uridines are converted to dihydrouridine, pseudouridine, ribothymidine residues

Question 12

Explain how alternative splicing may cause disease

Give two examples

Answer 12

1. Cis effects: mutation removal or addition of alternative splicing sites
2. Trans effects: Mutation affects expression of spliceosome components or regulatory proteins

—Retinitis pigmentosa and Spinal muscular atrophy are the results of mutations that disrupt the assembly or function of the snRNPs

Question 13

Describe RNA decay

What are the functions and the steps to RNA decay?

Answer 13

Cellular process that eliminates incorrectly processed RNA

Promotes ribonucleotide turnover

Can occur during transcription, as well as post-transcriptionally

Degrades tRNA and rRNA, which occurs primarily as a stress response or to eliminate a defective transcript

mRNA decay is an important mechanism to control protein production

Steps
1. Deadenylation
2. Decapping
3. 3’-5’ decay and 5’-3’ decay

Question 14

Which of the following is false?

1. The removal of spliceosomal introns requires spliceosomes that are complexes of ssRNAs and snRNPs.
2. Group Il introns require an exogenous nucleoside to initiate the cleavage reaction.
3. Both 5’-cap and 3’-poly stabilize and protect mRNAs, and aid in the export from the nucleus.
4. Alternative splicing is very important to protein diversity in higher organisms.

Answer 14

2. Group Il introns require an exogenous nucleoside to initiate the cleavage reaction.

—> Group I introns require an exogenous nucleoside. Group II introns require adenosine (A) at the branch site

Question 15

Is it possible that the top strand of the DNA is a template strand?

Answer 15

Yes, then the bottom strand would become the coding strand

Question 16

DNA template vs DNA coding strand

What’s the difference?

Answer 16

DNA template strand – serves as template for RNA polymerase

DNA coding strand – the non-template strand; has the same sequence as the RNA transcript (T in DNA is replaced with U in RNA)

Question 17

What is a promoter sequence?

In prokaryotes…

Answer 17

The promoter sequence is a specific DNA sequence located upstream of the coding sequence and contains binding sites for RNA polymerase and transcription factors.

The site on the DNA from which the first RNA nucleotide is transcribed is called the plus 1 site, or the initiation site.

Question 18

What is the function of RNA polymerase?

Answer 18

Carries out RNA synthesis using NTPs from a DNA sequence

Question 19

Give a general overview of RNA synthesis

Answer 19

One strand of DNA is used as a template for synthesis of a complementary strand via RNA polymerase.

Occurs in the 5′ to 3′ direction; No primer needed; Mg2+ is a cofactor.

The growing end of new RNA temporarily base-pairs with DNA template for ~8 bp.

The RNA polymerase generates positive supercoils ahead, later relieved by topoisomerases.

Lacks 3’ to 5’ exonuclease activity.

Mg2+ is used in both RNA and DNA synthesis as it helps to stabilize the

Question 20

Describe Footprinting technique to identify a protein-binding site on a DNA molecule

Answer 20

Premise: DNA bound by protein will be protected from chemical cleavage at its binding site

1. Isolate a DNA fragment thought to contain a binding site.
2. Radiolabel the DNA.
3. Bind protein to DNA in one tube; keep another as a “naked DNA” control.
4. Treat both samples with chemical or enzymatic agent to cleave the DNA (DNase I).
5. Separate the fragments by gel electrophoresis and visualize bands on X-ray film or imager plate

Question 21

Explain Sigma factors

Answer 21

Sigma factors are transcriptional factors that bind to specific DNA sequences

Target RNA polymerase

Primary Sigma factors responsible for initiating transcription of genes required for essential biochemical processes

Bacteria contain a variable number of related sigma factor proteins that activate the transcription of specific genes in response to growth signals or stress conditions

Question 22

Describe Dnase I footprinting

Answer 22

Laboratory technique to identify gene promoter sequences

Dnase I cleaves the phosphodiester bond on both strands, but does not cleave DNA where it binds to a protein

Utilizes autoradiography to visualize signals

Smallest fragment is on the top in autoradiography

Question 23

Describe EMSA

Electrophoretic Mobility Shift Assay

Answer 23

Used to detect DNA binding proteins

Gel-shift or band-shift assay

Useful for quantitative analysis of DNA binding proteins

Protein fractions are separated by column chormatography

Column fractions are incubated with a labeled probe

Samples are electrophoresed under conditions that preserve protein DNA interactions

Free probe not bound to protein migrates to the bottom of the gel

Question 24

What are the three phases of prokaryotic transcription?

Answer 24

1. Initiation: binding of RNA polymerase and the sigma factor
2. Elongation: dissociation of the sigma factor from RNA polymerase complex; mg2+ involved, DNA-RNA helix separates
3. Termination: Rho-dependent or Rho-independent

Rho: ATP-dependent helicase that binds to the newly synthesized RNA chain at a C-rich sequence

Question 25

Closed vs. Open RNA Polymerase Complex

Answer 25

Upon binding to the –35 and –10 boxes, the closed promoter complex transitions to an open complex, which initiates transcription and the release of the sigma factor. Transcription elongation then proceeds, beginning at the +1 site.

Open complex enters the transition from initiation to elongation during RNA synthesis

Closed complex (inactive) and open (active) complex

Question 26

Explain Prokaryotic Termination

Rho-dependent or Rho-independent

Answer 26

Rho-dependent
— ATP-dependent helicase destabilizes the RNA-DNA
— Requires both cis and trans factors
— Rho protein binds to C-rich region of new RNA

Rho-independent
— Does not require any trans-acting factors
— Depends on a GC-rich region followed by an A-rich region in DNA
— GC stem-loop causes pause in polymeration and dA:rU duplex dissociates and transcription is terminated

Rho protein is ATP-dependent helicase

Question 27

Describe Eukaryotic Promoter Sequences

Answer 27

RNA polymerase II promoter regions have an upstream TATA box sequence. The abbreviations Py2 and Py5 refer to a tract of two or five pyrimidine residues (C or T), respectively.

Question 28

Explain the 3 Eukaryotic RNA polymerases

Answer 28

RNA polymerase I synthesizes pre-ribosomal RNA (precursor for 28S, 18S, and 5.8S rRNAs).

RNA polymerase II is responsible for synthesis of messenger RNA (mRNA).
— very fast (500–1000 nucleotides/sec)
— specifically inhibited by mushroom toxin α-amanitin
— can recognize thousands of promoters
— large complex of 12 subunits
— has carboxy-terminal domain of highly conserved repeats

RNA polymerase III makes tRNAs, 5S rRNA, and some small RNA products.

Question 29

Explain Eukaryotic Transcription

Answer 29

1. Initiation: multiple transcription factors associate to the DNA template
2. Elongation: CTD region remains hyperphosphorylated
3. Termination: DNA template and trans-acting factors have signals. Termination is coupled with processing 3’ end poly A tail of mRNA.Pol II is dephosphorylated. It is coupled with processing of the 3’ end of mRNA.

–> A functional poly(A) signal is generally required for efficient termination of mRNA synthesis in eukaryotes.

Prokaryotic transcription: the poly A tail added after transcription

Question 30

Describe the Assembly of RNA polymerase II at the promoter

Answer 30

Initiated by TATA-binding protein (TBP) with the promoter important to recruit RNA polymerase II
— TBP is part of multisubunit complex TFIID.
—– Other proteins include TFIIB, TFIIA, TFIIF, TFIIE and TFIIH.

Helicase activity in TFIIH unwinds DNA at the promoter.

Kinase activity in TFIIH phosphorylates the polymerase at the CTD (carboxy-terminal domain), changing the conformation and enabling RNA Pol II to transcribe.

Pol II has 12 subunits and catylizes mRNA synthesis

Question 31

What are the functions of TBP and TFIIH?

Answer 31

TBP: TATA-binding protein specifically recognizes the TATA box

TFIIH: unwinds DNA at the promoter via helicase activity; phosphorylates Pol II within the CTD; Recruits nucleotide-excision repair proteins

Question 32

Which of the following is False?

1. RNA polymerases don’t need primers to initiate RNA synthesis.
   
2. Sigma factor binds to -10 and -35 boxes in prokaryotes.
   
3. RNA polymerases don’t need magnesium for RNA synthesis.
   
4. TATA-binding protein is required for RNA polymerase Il to bind to the promoter sequences in eukaryotes.

Answer 32

3. RNA polymerases don’t need magnesium for RNA synthesis.

Magnesium ions (Mg2+) are required for both RNA and DNA synthesis because they are used to help stabilize the RNA/DNA strands during synthesis

Question 33

What are 3 ways in which RNA may be considered a dynamic biomolecule?

Answer 33

1. RNA rapid turnover
2. RNA forms tertiary structure that is altered by the binding of ligands
3. RNA can bind to RNA, genomic DNA and protein to regulate gene expression

Question 34

What are three forms of RNA?

Answer 34

Messenger RNAs (mRNA) encode the amino acid sequences of all the polypeptides found in the cell.

Transfer RNAs (tRNA) match their anticodon to the mRNA while carrying a specific amino acid used for protein synthesis.

Ribosomal RNAs (rRNA) are constituents of the large and small ribosomal subunits.

Question 35

Name 3 “lesser understood” functions of RNA in eukaryotic cells

Answer 35

Messenger RNAs (mRNA) encode the amino acid sequences of all the polypeptides found in the cell.

Transfer RNAs (tRNA) match their anticodon to the mRNA while carrying a specific amino acid used for protein synthesis.

Ribosomal RNAs (rRNA) are constituents of the large and small ribosomal subunits.

Question 36

What is the connection between RNA and viruses?

Answer 36

RNA molecules act as genomic material

Question 37

What are the 5 categories of Long noncoding RNAs (lncRNAs)?”

Answer 37

1. Sense - The lncRNA sequence overlaps with the sense strand of a protein coding gene.
2. Antisense - The lncRNA sequence overlaps with the antisense strand of a protein coding gene.
3. Bidirectional - The lncRNA sequence is located on the opposite strand from a protein coding gene whose transcription is initiated less than 1000 base pairs away.
4. Intronic - The lncRNA sequence is derived entirely from within an intron of another transcript. This may be either a true independent transcript or a product of pre-mRNA processing
5. Intergenic - The lncRNA sequence is located between two protein coding genes, but not near any other protein coding loci

Question 38

Is the expression of many genes on X-chromosome 2-fold higher in female than in male?

Answer 38

No because of the XIST gene.

XIST: x-inactive specific transcript. The XIST gene on the human X chromosome is transcribed into an lncRNA that regulates the process of X inactivation.

Question 39

Describe ribozymes

Answer 39

Cleave themselves or another RNA
3-D structure integral to function

Inactive if denatured or if essential nucleotides changed

Small nucleolytic ribozymes undergo transesterification reactions in cis, in which a specific 2′-hydroxyl attacks the neighbouring 3′,5′-phosphodiester bond. This results in a 2′,3′-cyclic phosphate and a 5′ hydroxyl at the 3′ cleavage products[1]

Example: RNase P cleaves precursors to tRNAs by recognizing the shape of the pre-tRNA and the CCA sequences

Question 39

How do prokaryotic tRNA and rRNA differ from their eukaryotic versions?

Answer 39

In prokaryotes, tRNA and rRNA molecules require the removal of spacer sequences to generate the mature forms of tRNA and rRNA.

Question 40

Which of the following is false?

1. tRNAs are required for protein synthesis.

2. shoRNAs are involved in rRNA processing.

3. The expression of genes on X-chromosome is 2-fold higher in females than in males.

4. Some RNA molecules can act as enzymes that can hydrolyze RNA molecules.

Answer 40

3. The expression of genes on X-chromosome is 2-fold higher in females than in males.

XIST

Question 41

Which factor is NOT involved in mRNA processing in vertebrates?

• the nucleoleus
• ribonucleases
• 30S pre-rRNA
• snoRNAs
• snoRNPs

Answer 41

• 30S pre-rRNA

Question 42

Which statement about E.coliRNA polymerase is FALSE?

• RNA polymerase holoenzyme has several subunits.
• Core enzyme selectively binds promoter regions but cannot initiate synthesis without a sigma factor.
• RNA produced by this enzyme will be completely complementary to the DNA template.
• The enzyme adds nucleotides to the 3’end of the growing RNA chain.
• The enzyme cannot synthesize RNA in the absence of DNA.

Answer 42

• Core enzyme selectively binds promoter regions but cannot initiate synthesis without a sigma factor.

Question 43

The sigma factor of E. coli RNA polymerase…

• will catalyze synthesis of RNA from both DNA template strands in the absence of the core enzyme.
• is inseparable from the core enzyme.
• is required for termination of an RNA chain.
• associates with the promoter before binding core enzyme.
• combines with the core enzyme to confer specific binding to a promoter

Answer 43

Combines with the core enzyme to confer specific binding to a promoter