Biochem II Ch. 26: RNA Metabolism

*Prokaryotic* Where are the consensus sequences in E.coli RNA polymerase?

-10 and -35

*Eukaryotic* Where do we find the TATA box?

-30

*Prokaryotic* Where does the promoter region for RNA polymerase extend?

-70 to +30

How is the 5' cap formed?

-condensation of a molecule of GTP with the triphosphate at the 5' end of the transcript -forms a 5'-5' triphosphate linkage

What are the eukaryotic RNA polymerases and what are their functions?

1. RNA Pol I: synthesizes preribosomal RNA 2. RNA Pol II: synthesizes mRNA 3. RNA Pol III: synthesizes tRNA and one of the preribosmal RNA

*Eukaryotic* What are the steps involved in the ordered assembly of the TFII complex?

1. TATA binding protein (TBP) recognizes TATA box 2. TBP is bound by TFIIB which binds DNA on either side of TBP 3. TFIIA binds with TFIIB to stabilize TBP-DNA complex 4. TFIIB-TBP complex is bound by TFIIF/Pol II complex 5. TFIIE + TFIIH bind creating the a closed complex

Describe the steps in the sigma cycle

1. The RNA pol binds to the promotor by the direction of the sigma factor 2. The subunit dissociates as elongation occurs 3. NusA protein competes with sigma by binding to elongating RNA polymerase 4. NusA dissociates at the completion of transcription 5. RNA dissociates from DNA 6. sigma binds to reinitiate transcription

What are the basic steps in splicing to create a mature mRNA

1. get rid of the introns 2. cap the 5' end 3. create a poly(A) tail

What are the three major kinds of RNA that are produced during transcription?

1. mRNA 2. tRNA 3. rRNA

What are the basic steps in footprinting?

1. radiolabel one strand 2. introduce random breaks 3. separate the cleavage products by electrophoresis

What are the two classes of termination signals in E.coli?

1. rho dependent 2. rho independent

What are the two roles of complementary DNA strands in transcription?

1. template strand: serves as RNA template 2. coding strand: is identical in base sequence to the RNA transcribed from the gene, but with U in place of T.

RNA polymerase requires what things?

1.DNA template 2. ATP, GTP, CTP, UTP 3. Mg2+

*Eukaryotic* How many subunits does RNA Pol II have?

12

RNA is built in what direction?

5'-3'

The transcription bubble of RNA has how many base pairs of RNA:DNA

8

Which of the following statements about the synthesis of rRNA and tRNA in E. coli is true? A) Both rRNA and some tRNAs are part of the same primary transcript. B) Each rRNA sequence (16S, 23S, 5S) is transcribed into a separate primary transcript. C) Primary tRNA transcripts undergo methylation, but rRNA sequences are not methylated. D) The tRNA sequences all lie at the 3'end of the rRNA transcripts E) There is a single copy of the rRNA genes.

A) Both rRNA and some tRNAs are part of the same primary transcript.

Which of the following statements about E. coli RNA polymerase is false? A) Core enzyme selectively binds promoter regions, but cannot initiate synthesis without a sigma factor. B) RNA polymerase holoenzyme has several subunits. C) RNA produced by this enzyme will be completely complementary to the DNA template. D) The enzyme adds nucleotides to the 3' end of the growing RNA chain. E) The enzyme cannot synthesize RNA in the absence of DNA

A) Core enzyme selectively binds promoter regions, but cannot initiate synthesis without a sigma factor.

Which of the following is not known to be involved in initiation by eukaryotic RNA polymerase II? A) DNA helicase activity B) DNA polymerase activity C) Formation of an open complex D) Protein binding to specific DNA sequences E) Protein phosphorylation

B) DNA polymerase activity

Which one of the following is not true of the mRNA for ovalbumin? A) Exons are used for polypeptide synthesis. B) Introns are complementary to their adjacent exons and will form hybrids with them. C) The mature mRNA is substantially shorter than the corresponding region on the DNA. D) The mRNA is originally synthesized in the nucleus, but ends up in the cytoplasm. E) The splicing that yields a mature mRNA occurs at very specific sites in the RNA primary transcript.

B) Introns are complementary to their adjacent exons and will form hybrids with them.

Which one of the following statements about E. coli RNA polymerase (core enzyme) is false? A) It can start new chains de novo or elongate old ones. B) It has no catalytic activity unless the sigma factor is bound. C) It uses nucleoside 5'-triphosphates as substrates. D) Its activity is blocked by rifampicin. E) Its RNA product will hybridize with the DNA template.

B) It has no catalytic activity unless the sigma factor is bound.

Which one of the following statements about eukaryotic RNA polymerases is correct? A) All three eukaryotic RNA polymerases recognize the same promoters as prokaryotic polymerases. B) None of the eukaryotic RNA polymerases recognizes prokaryotic promoters. C) Only eukaryotic RNA polymerase I recognizes prokaryotic promoters. D) Only eukaryotic RNA polymerase II recognizes prokaryotic promoters. E) Only eukaryotic RNA polymerase III recognizes prokaryotic promoters.

B) None of the eukaryotic RNA polymerases recognizes prokaryotic promoters.

The excision (splicing) of many group I introns requires, in addition to the primary transcript RNA: A) a cytosine nucleoside or nucleotide and a protein enzyme. B) a guanine nucleoside or nucleotide (only). C) a protein enzyme only. D) a small nuclear RNA and a protein enzyme. E) ATP, NAD, and a protein enzyme.

B) a guanine nucleoside or nucleotide (only).

RNA polymerase: A) binds tightly to a region of DNA thousands of base pairs away from the DNA to be transcribed. B) can synthesize RNA chains de novo (without a primer). C) has a subunit called λ (lambda), which acts as a proofreading ribonuclease. D) separates DNA strands throughout a long region of DNA (up to thousands of base pairs), then copies one of them. E) synthesizes RNA chains in the 3' → 5' direction.

B) can synthesize RNA chains de novo (without a primer).

After binding by E. coli RNA polymerase, the correct order of events for transcription initiation is: A) closed complex formation, open complex formation, promoter clearance, start of RNA synthesis. B) closed complex formation, open complex formation, start of RNA synthesis, promoter clearance. C) open complex formation, closed complex formation, start of RNA synthesis, promoter clearance. D) start of RNA synthesis, closed complex formation, open complex formation, promoter clearance. E) start of RNA synthesis, open complex formation, closed complex formation, promoter clearance

B) closed complex formation, open complex formation, start of RNA synthesis, promoter clearance.

The sigma factor of E. coli RNA polymerase: A) associates with the promoter before binding core enzyme. B) combines with the core enzyme to confer specific binding to a promoter. C) is inseparable from the core enzyme. D) is required for termination of an RNA chain. E) will catalyze synthesis of RNA from both DNA template strands in the absence of the core enzyme.

B) combines with the core enzyme to confer specific binding to a promoter.

Processing of a primary mRNA transcript in a eukaryotic cell does not normally involve: A) attachment of a long poly(A) sequence at the 3' end. B) conversion of normal bases to modified bases, such as inosine and pseudouridine. C) excision of intervening sequences (introns). D) joining of exons. E) methylation of one or more guanine nucleotides at the 5' end

B) conversion of normal bases to modified bases, such as inosine and pseudouridine.

The 5'-terminal cap structure of eukaryotic mRNAs is a(n): A) 7-methylcytosine joined to the mRNA via a 2',3'-cyclic linkage. B) 7-methylguanosine joined to the mRNA via a 5' → 3' diphosphate linkage. C) 7-methylguanosine joined to the mRNA via a 5' → 5' triphosphate linkage. D) N6-methyladenosine joined to the mRNA via a 5' → 5' phosphodiester bond. E) O6-methylguanosine joined to the mRNA via a 5' → 5' triphosphate linkage.

C) 7-methylguanosine joined to the mRNA via a 5' → 5' triphosphate linkage.

Which of the following is not usually essential for the catalytic activity of ribozymes? A) Correct base pairing B) Correct base sequence C) Correct interaction with protein D) Correct secondary structure E) Correct three-dimensional structure

C) Correct interaction with protein

Which of the following statements about E. coli RNA polymerase (core enzyme) is false? A) In the absence of the σ subunit, core polymerase has little specificity for where initiation begins. B) The core enzyme contains several different subunits. C) The core enzyme has no polymerizing activity until the σ subunit is bound. D) The RNA chain grows in a 5' → 3' direction. E) The RNA product is complementary to the DNA template.

C) The core enzyme has no polymerizing activity until the σ subunit is bound.

*Eukaryotic* At what terminal do we find RPB1?

C-terminal

Which one of the following properties of the L-19 IVS ribozyme is not shared with enzymes that are purely protein? A) It acts as a true catalyst. B) It can be competitively inhibited. C) It displays Michaelis-Menten kinetics. D) It exploits base-pairing with internal guide sequences. E) It makes use of covalent and metal ion catalysis.

D) It exploits base-pairing with internal guide sequences.

RNA polymerase from E. coli (core enzyme alone) has all of the following properties except that it: A) can extend an RNA chain and initiate a new chain. B) is required for the synthesis of mRNA, rRNA, and tRNA in E. coli. C) produces an RNA polymer that begins with a 5'-triphosphate. D) recognizes specific start signals in DNA. E) requires all four ribonucleoside triphosphates and a DNA template.

D) recognizes specific start signals in DNA.

RNA polymerase is directed by

DNA

The polymerase used in transcription is

DNA-dependent RNA polymerase

Which one of the following statements about mRNA stability is true? A) Degradation always proceeds in the 5' to 3' direction. B) Degradation of mRNA by polynucleotide phosphorylase yields 5'-nucleoside monophosphates. C) In general, bacterial mRNAs have longer half-lives than do eukaryotic mRNAs. D) Rates of mRNA degradation ared always at least 10-fold slower than rates of mRNA synthesis. E) Secondary structure in mRNA (hairpins, for example) slows the rate of degradation

E) Secondary structure in mRNA (hairpins, for example) slows the rate of degradation

Splicing of introns in nuclear mRNA primary transcripts requires: A) a guanine nucleoside or nucleotide. B) endoribonucleases. C) polynucleotide phosphorylase. D) RNA polymerase II. E) small nuclear ribonucleoproteins (snurps).

E) small nuclear ribonucleoproteins (snurps).

A branched ("lariat") structure is formed during: A) attachment of a 5' cap to mRNA. B) attachment of poly(A) tails to mRNA. C) processing of preribosomal RNA. D) splicing of all classes of introns. E) splicing of group II introns.

E) splicing of group II introns.

Differential RNA processing may result in: A) a shift in the ratio of mRNA produced from two adjacent genes. B) attachment of the poly(A) tail to the 5' end of an mRNA. C) inversion of certain exons in the final mRNA. D) the production of the same protein from two different genes. E) the production of two distinct proteins from a single gene.

E) the production of two distinct proteins from a single gene.

"Footprinting" or DNase protection is a technique used to identify: A) a region of DNA that has been damaged by mutation. B) E. coli cells that contain a desired, cloned piece of DNA. C) the position of a particular gene of a chromosome. D) the position of internally double-stranded regions in a single-stranded DNA molecule. E) the specific binding site of a repressor, polymerase, or other protein on the DNA.

E) the specific binding site of a repressor, polymerase, or other protein on the DNA.

A technique derived from principles used in DNA sequencing. Allows one to identify the DNA sequences bound by a particular protein.

Footprinting

What stops the sigma factor from binding again?

NusA protein binds competitively with sigma factor

*Eukaryotic* Required at every Pol II promotor. Similar in function to sigma factors in bacteria.

Pol II transcription factors (TFII)

What causes the gap in the DNA ladder in the presence of protein?

Protein shows where binding occurs. It is where DNA binding protein was present. When the protein is present, it prevents cleavage. The labeled sites that terminate in binding sites are gone.

We make DNA processive by using the β-clamp, how do we make RNA processive?

RNA is already clamped and ready to go

The polymerase that is central to eukaryotic gene expression

RNA pol II

*Eukaryotic* What is the largest subunit in RNA Pol II and what prokaryotic subunit is it most similar to?

RPB1 β'

*Eukaryotic* What subunit is similar to the β subunit in prokaryotes?

RPB2

*Eukaryotic* What subunits show structural homology to the two bacterial α subunits?

RPB3 RPB11

T/F Regulation can occur at any step

T

*Eukaryotic* Major assembly point for the preinitiation complexes of Pol II

TATA box

Protein can _______ transcription or ____ it

activate repress

The elongation of RNA strands by RNA polymerase in both bacteria and in eukaryotes is inhibited by

antibiotic actinomycin D

When does the sigma factor dissociate?

at the beginning of elongation

What is one protein that activates transcription in E.coli?

cAMP receptor protein (CRP)

Coding segments are called

exons

*Prokaryotic* What is the role of sigma in prokaryotic RNA polymerase

guides the polymerase to the associated DNA promotor sequence

What are the activities of TFIIH?

helicase: unwind DNA at promoter kinase: phosphorylates Pol II recruits nucleotide excision repair proteins

What does CRP do?

increases transcription of genes coding for enzymes that metabolize sugars

Noncoding tracts that break up the coding region of the transcript are called

introns

*Prokaryotic* Describe the structure of DNA-dependent RNA polymerase in E.coli

large complex five core subunits a sixth subunit designated as sigma

What do we call a newly synthesized RNA molecule?

primary transcript

What is the function of the 5' cap?

protects mRNA from ribonuclease binds to a specific cap-binding complex of proteins

Proteins that block the synthesis of RNA at specific genes

repressors

How are rho dependent and rho independent terminating transcription?

rho independent relies on a terminator sequence. rho dependent utilizes rho helicase

What is the transcription initiation factor in prokaryotes?

sigma factor

What do we call the process in which introns are removed from the primary transcript and the exons are joined to form a continuous sequence?

splicing

Conversion of genetic information in a segment of double stranded DNA into an RNA strand

transcription

When does RNA polymerase bind to the promoters of heat shock genes?

when σ70 is replaced with σ32

What is the primary sigma factor?

σ70