MBC-05

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RNA Transcription and processing

True/False B-thalassemia is caused by only one point mutation.

False. There is a distribution of mutations throughout the B-globin gene and each can cause various severity B-thalassemia.

Define transcription.

The enzymatic process whereby the genetic information contained in one strand of DNA is used to specify a complementary sequence of bases in an mRNA chain.

What is the major structural difference between DNA and RNA?

The presence of a hydroxyl (-OH) group on the 2'-carbon of the ribose sugar. In DNA, this hydroxyl is absent. The hydroxyl is present in RNA making RNA more labile (more easily altered)

Is RNA usually characterized as single-stranded or double-stranded?

Single-stranded.

Can single-stranded RNA coil in specific secondary structures?

Yes, such as hairpin loops, and these are critical for some RNA functions like those of rRNA.

What are the 3 major types of RNA?

Messenger RNA (mRNA), Transfer RNA (tRNA), and Ribosomal RNA (rRNA).

Which type of RNA is produced during transcription and codes for proteins?

mRNA.

When the large RNA polymerase protein complex attaches to dsDNA, which strand is pushed out of the way, the non-template strand, or the template strand?

The non-template strand.

True/False RNA Polymerase does not need to unwind dsDNA for transcription.

False. RNA Polymerase locally unwinds DNA as it synthesizes RNA.

Is there significant primase activity early in transcription?

No. There is no primer needed for synthesis of mRNA.

What is the direction of synthesis of mRNA?

Like DNA synthesis, nucleotides are added to the 3' end of a growing mRNA strand. Thus, 5' to 3'.

What is the function of topoisomerase in transcription?

Topoisomerase relaxes the supercoiling that occurs due to local unwinding of dsDNA.

True/false: the standard notation of RNA and DNA is to ALWAYS write 5' to 3'.

True. If the orientation is not indicated, assume it is 5' to 3'.

Which strand of dsDNA is in physical contact with the RNA transcript, the template strand, or the non-template strand?

The template strand.

Which strand of DNA is also called the "coding" strand, the template strand or the non-template strand?

The non-template strand is the coding strand.

Why is the coding strand called the coding strand?

Because it is very similar to the RNA transcript. In fact, it is identical except that T is replaced by U.

What transcript comes from the following coding strand?

(5') CGCTAT (3')

(5') CGCUAU (3')

Regarding RNA polymerase, what is a "holoenzyme"

An enzyme with multiple protein subunits, the complete complex containing all of the subunits needed for activity.

What is the function of the sigma subunit of E. coli RNA polymerase?

To bind to the promoter region.

What is the function of the 2 beta subunits of E. coli RNA polymerase?

To form the active site of RNA synthesis.

If RNA polymerase does not need a primer to start transcription (like DNA polymerase does for replication), then how does the polymerase "know" where to start transcribing?

RNA polymerase binds to a gene's promoter region, importantly, to the consensus sequences.

What are the two important upstream (5') regions of the promoter sequence in prokaryotes such as E. coli?

The TATAAT "box" in the -10 region, and the TTGACA "box" in the -35 region.

What subunit of E. coli RNA polymerase binds to the -10 and -30 promoter elements?

The sigma subunit.

In prokaryotes, What are the five important first steps for RNA polymerase to initiate synthesis of an RNA transcript and move into elongation?

1. RNA polymerase holoenzyme binds relatively weakly to the DNA to form a "closed complex". 2. The sigma subunit binds to promoter elements. 3. The holoenzyme forms a more tightly bound "open complex" in which about 10 bp of the dsDNA are locally opened. 4. The sigma subunit falls off and RNA polymerase is stably associated with DNA, 5. elongating the transcript processively.

In prokaryotes, how is transcription terminated independent of Rho?

Conserved bases in the DNA contain a palindrome sequence and a PolyA sequnce. The RNA transcript of these are a palindrome forming a hairpin loop, and PolyU stem. The stem and loop terminate transcription.

How does Rho terminate transcription in prokaryotes?

Less well understood. The multi-subunit protein Rho binds to the Rut sequence of RNA, then travels along the nascent RNA until it encounters the RNA polymerase, where it disrupts transcription.

Which eukaryotic RNA polymerase synthesizes pre-ribosomal RNA, the precursor for 18S, 5.8S and 28S RNA?

Pol I.

Which eukaryotic RNA polymerase synthesizes mRNA and some specialized RNAs?

Pol II.

Which eukaryotic RNA polymerase synthesizes tRNA, 5S RNA and other small specialized RNAs?

Pol III.

What are the two important promoter consensus sequences in eukaryotes?

The TATA box located at -30, and the invariant sequence located at +1.

Prokaryotic or Eukaryotic transcription? The RNA polymerase directly binds to the DNA template.

Prokaryotic.

Prokaryotic or Eukaryotic? The RNA polymerase binds to a cascade of proteins including the TBPs (TATA binding proteins) to connect to the DNA template.

Eukaryotic.

Describe the function of Actinomycin D.

Inhibits RNA elongation by intercalating into DNA template. Works on prokaryotes and eukaryotes.

Describe the function of Acridine.

Inhibits RNA elongation by intercalating into DNA template. Works on prokaryotes and eukaryotes.

Describe the function of Rifampicin.

Inhibits bacterial RNA synthesis by binding to the beta-subunit of the polymerase. Good for treating TB.

Describe the function of alpha-amanitin.

Blocks Pol II and Pol III (higher concentrations) but NOT Pol I or bacterial RNA polymerase. Found in some wild mushrooms, causes severe clinical problems.

When it comes to RNA processing, are we generally referring to eukaryotic or prokaryotic mRNA, or both?

Generally, eukaryotic RNA is the heavily processed type. There are exceptions.

What is the first step of RNA processing? When does it occur?

Addition of a 5' cap, occurs during transcription to protect the labile 5' end from degradation.

True/False: The first transcript is called "primary" "immature" and/or "hnRNA (heteronuclear)".

True.

What are the three major aspects of RNA processing?

Addition of a 5' cap, excision of introns, and addition of a PolyA tail.

True/False: the addition of a 5' cap is performed by one enzyme, 2'-O-methyltransferase.

False. There are multiple enzymes and steps required. The enzyme listed is the final one.

Does the addition of a 5' cap require energy?

Yes, in the form of GTP.

What is the chemical structure of the 5' cap?

7-methyl-guanosine.

What is the unique connection between the ribose sugars of the 5' cap and the end of the transcript?`

A 5' to 5' linkage.

What are the steps in the addition of a PolyA tail?

An enzyme complex binds to a cleavage signal sequence (AAUAA) and displaces RNA polymerase. An endonuclease cleave the RNA 3' of the cleavage signal sequence. Polyadenylate polymerase synthesizes the PolyA tail.

What enzyme is responsible for the addition of the PolyA tail?

Polyadenylate polymerase.

True/False. The number of A's in the PolyA tail can vary.

True. More A's means more stability.

Is the primary type of introns found in mRNA transcripts self-splicing or non-self splicing?

Non-self splicing.

What is the function of snRNPs (small nuclear ribonucleoprotein particles: RNA+protein)?

Splicing introns.

What is systemic lupus erythematosus?

An autoimmune disease in which the patient produces antibodies against snRNPs. Fatal.

What are the 2 steps of intron splicing?

First, cleavage occurs at the 5' splice site (splice donor site) with joining of the 5' end of the intron to an A (Branch site/branch point) within the intron. Second, cleavage occurs at the 3' splice site (splice acceptor site) with ligation of the exons.

What is the shape of the intron after it is spliced?

Lariat.

What is a spliceosome?

An RNA/protein complex almost as large as a ribosome, made of five 100-300 base snRNAs (U1, U2, U4, U5, U6) and their interacting proteins. Splices introns from RNA transcripts.

What are the three primary functions of snRNPs

Recognize sites (splice site and branch point site)
Bring these sites together
Catalyze cleavage reactions that involve the sites

True/False: The complex secondary structure of an snRNP allows proteins to surround it and have it function. Part of the snRNP is complementary to the 5' splice site (aka the donor site)

True.

True/False: Splicing requires several snRNPs (various U's, i.e. U1, U2, etc.) with various functions.

True.

Which of the following is not required for transcription? DNA template, DNA-dependent RNA polymerase, Primer, Nucleotides

Primer.

True/False: Topoisomerases relax supercoiling of DNA brought about by transcription.

True.

True/False: The RNA transcript is synthesized from the DNA coding strand.

False.

True/False: Prokaryotes have three kinds of nuclear RNA polymerases whereas eukaryotes have only one.

False.

Consensus sequences have not been identified for DNA promoter regions.

False.

True/False: Eukaryotic primary transcripts may contain introns.

True.

True/False: The most extensive processing of primary transcripts occurs in eukaryotic mRNAs and in tRNAs of both bacteria and eukaryotes.

True.

True/False: Ribozymes are catalytic RNAs that process RNA.

True.

True/False: Ribosomal RNAs are the most abundant type of RNA in the cell and are constituents of ribosomes.

True.

True/False: Messenger RNAs are the most abundant type of RNA in the cell.

False.

True/False: Transfer RNAs encode the amino acid sequence of one or more polypeptides.

False. mRNAs do this.

True/False: Exons do not code for amino acids.

False.

True/False: The primary mRNA transcript is also known as hnRNA (heterogeneous nuclear RNA).

True.

True/False: Introns are intervening sequences found between regions of coding DNA.

True.

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