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BIOC 302: Molecular Biology > RNA Transcription and Processing P1 > Flashcards

RNA Transcription and Processing P1 Flashcards

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1
Q

What happens if transcription does not start at the correct spot on the DNA?

A

If transcription does not start at the correct spot, the desired protein will not be produced.

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1
Q

What is the primary purpose of RNA transcription?

A

RNA transcription transcribes the stable information encoded in DNA into a readable version that can be translated into a protein.

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2
Q

How does the template strand of DNA relate to the RNA produced during transcription?

A

The template strand of DNA is used to produce a complementary RNA strand, where uracil (U) is used instead of thymine (T), and ribose is used instead of deoxyribose.

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3
Q

What are some types of RNA produced by transcription besides coding RNA?

A

Non-coding RNA types produced by transcription include:

RNA for making ribosomes (rRNA)
Transfer RNA (tRNA) that bridges the message and peptide
Other non-coding RNAs

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4
Q

What is the role of RNA polymerase in transcription initiation?

A

RNA polymerase binds to promoter sequences, opens the DNA, and begins building the RNA strand by forming phosphodiester bonds.

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5
Q

What is the role of the RNA-DNA hybrid during transcription elongation?

A

During elongation, the growing RNA strand temporarily base pairs with the DNA template, forming a short RNA-DNA hybrid before the RNA is released.

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6
Q

How is RNA polymerase in transcription similar to DNA polymerase? (3)

A

Both enzymes:

Work in the 5’ to 3’ direction
Use magnesium as a co-factor
Catalyze phosphodiester bond formation powered by hydrolysis of the incoming nucleotide

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7
Q

How is RNA polymerase different from DNA polymerase? (3)

A

Differences include:

RNA polymerase does not need a primer
It lacks 3’ to 5’ exonuclease proofreading, making it more error-prone
It uses NTPs (ribonucleotides) instead of dNTPs

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8
Q

What is the role of the template and non-template strands during transcription?

A

The template strand serves as the guide for RNA synthesis, while the non-template strand (coding strand) is identical to the RNA, except that U replaces T in RNA.

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9
Q

What is the function of the σ subunit in the RNA polymerase holoenzyme?

A

The σ subunit directs RNA polymerase to specific DNA binding sites at promoter regions, ensuring correct initiation of transcription.

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10
Q

Why does RNA polymerase have a higher error rate than DNA polymerase?

A

RNA polymerase lacks a separate 3’ to 5’ exonuclease proofreading mechanism, resulting in a higher error rate, but transcription errors are more tolerated.

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11
Q

What role does the σ70 subunit play in RNA transcription?

A

The σ70 subunit binds to promoter regions of housekeeping genes and guides RNA polymerase to the correct start site of transcription.

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12
Q

How does the σ factor recognize promoter regions?

A

The σ factor interacts with specific sequences at the -35 and -10 regions upstream of the transcription start site, ensuring correct alignment of RNA polymerase.

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13
Q

What structural changes occur during transcription initiation?

A

RNA polymerase and the σ factor bind to form a closed complex, then a small region of DNA is separated, forming an open complex, allowing RNA transcription to begin.

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14
Q

How does RNA elongation proceed after initiation?

A

After the σ factor leaves the complex, the RNA exits through the RNA channel, and transcription continues until the end of the transcript.

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15
Q

What is the role of promoter sequences in transcription initiation?

A

Promoter sequences determine where RNA polymerase binds, regulating which sequences will be transcribed and determining the start of the RNA transcript.

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16
Q

Can transcription occur on both DNA strands at the same time?

A

Transcription can occur from both strands, but not simultaneously. Each strand can produce different RNA or protein sequences depending on the genes present.

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17
Q

What is RNA Polymerase II (Pol II) responsible for synthesizing?

A

RNA Polymerase II is responsible for synthesizing mRNAs and many non-coding RNAs (ncRNAs) in eukaryotic cells.

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18
Q

How does RNA Polymerase II regulate protein expression in the genome?

A

RNA Polymerase II recognizes thousands of promoters, and the varying strengths of these promoters help regulate how much protein is expressed in the genome.

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19
Q

What is the TATA box, and where is it located relative to the transcription start site?

A

The TATA box is a consensus sequence (TATA(A/T)A(A/T)(A/G)) found near the -30 position upstream of the transcription start site and is similar to the -10 element found in prokaryotes.

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20
Q

What role does the Inr sequence play in RNA transcription?

A

The Inr sequence (initiator) is located at the +1 position and helps direct the transcription machinery to the correct start site for RNA synthesis.

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21
Q

What proteins are required for the initiation of transcription at RNA Pol II promoters in eukaryotes?

A

RNA Pol II, TFII proteins (transcription factors), and kinases are required. Kinases phosphorylate the C-terminal domain (CTD) of RNA Pol II, marking the transcriptional progress.

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22
Q

How do TFII proteins contribute to transcription in eukaryotes?

A

TFII proteins are general transcription factors required for most transcription initiation and elongation, helping position Pol II at the correct site on the promoter.

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23
Q

What is the function of the protein mediator in RNA Pol II transcription?

A

The protein mediator regulates transcription by driving specific activation or repression of RNA Pol II, providing fine control at specific gene loci.

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24
Q

What structural change helps initiate transcription at Pol II promoters?

A

Significant bending of the promoter element opens the DNA at these regions, creating a transcription bubble and allowing RNA Pol II to begin transcription.

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25
Q

What happens after the initiation factors are released during transcription?

A

After initiation factors dissociate, RNA elongation proceeds as the RNA exits the polymerase, and the C-terminal domain becomes phosphorylated, driving the process forward.

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26
Q

How does phosphorylation of the CTD of RNA Pol II regulate transcription?

A

Phosphorylation of the CTD creates platforms for different proteins to bind, triggering transitions between transcriptional phases, such as initiation and elongation.

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27
Q

How does TFIID position TBP and Pol II at the promoter?

A

TFIID binds the promoter DNA in an elongated complex, anchoring TBP-DNA interactions and ensuring that Pol II is positioned at the correct distance for transcription initiation.

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28
Q

What role do chromatin remodeling proteins play in transcription initiation?

A

Chromatin remodeling proteins use ATP to move nucleosomes out of the way, allowing the transcription machinery to access the DNA and initiate transcription.

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29
Q

What triggers elongation during transcription?

A

Elongation is triggered by a shift in the phosphorylation pattern along the CTD of Pol II, enabling RNA synthesis to proceed through the gene.

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30
Q

How does the phosphorylation status of the CTD influence transcription termination?

A

As the transcription complex approaches the end of the gene, dephosphorylation of the CTD leads to the release of the RNA and termination of transcription.

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31
Q

How are transcription factories visualized in eukaryotic cells?

A

In immunofluorescence experiments, transcription factories are seen as primed regions in the genome, where the transcription machinery is assembled and ready for activation upon receiving the correct phosphorylation signals.

32
Q

What is the primary role of RNA Polymerase II (Pol II) in eukaryotic transcription?

A

RNA Polymerase II is responsible for the synthesis of mRNAs and many non-coding RNAs in eukaryotic cells.

33
Q

How does RNA Polymerase II regulate gene expression at the level of promoters?

A

RNA Polymerase II can recognize thousands of different promoters with varying strengths, allowing it to regulate how much protein is expressed by controlling transcription initiation.

34
Q

What are two common sequence elements found in some RNA Polymerase II promoters?

A

The TATA box, a consensus sequence near -30, and the Inr (initiator) sequence at +1.

35
Q

What role do transcription factors (TFII proteins) play in RNA Polymerase II initiation?

A

TFII proteins are required for initiating and elongating transcription by RNA Polymerase II. They help position the polymerase and phosphorylate the C-terminal domain (CTD) to facilitate transcription.

36
Q

Describe the significance of the C-terminal domain (CTD) of RNA Polymerase II.

A

The CTD is a flexible region with repeated sequences that can be phosphorylated at various stages of transcription. Phosphorylation of the CTD helps regulate elongation and termination of transcription.

37
Q

How does chromatin structure impact transcription initiation in eukaryotes?

A

Chromatin must be remodeled to allow transcription. ATP-dependent chromatin remodeling proteins help move nucleosomes out of the way so that RNA Polymerase II can access the DNA.

38
Q

What is the role of the transcription factor TFIID in RNA Polymerase II-mediated transcription?

A

TFIID binds to the promoter DNA and helps position the TBP (TATA-binding protein) and RNA Polymerase II over the transcription start site, facilitating transcription initiation.

39
Q

What structural change in DNA is necessary for transcription initiation by RNA Polymerase II?

A

Significant bending of the promoter element is required to help open the DNA, creating a transcription bubble for the RNA-DNA duplex to form.

40
Q

How is the elongation of transcription driven in RNA Polymerase II?

A

Elongation is driven by the release of initiation factors and phosphorylation of the CTD, allowing RNA Polymerase II to move along the DNA and synthesize RNA.

41
Q

What is the role of phosphorylation in the regulation of RNA Polymerase II transcription?

A

Phosphorylation of the CTD signals different stages of transcription, including elongation, processing, and termination. Shifts in phosphorylation status at different stages regulate the activity of RNA Polymerase II.

42
Q

What happens to the initiation factors after RNA Polymerase II moves beyond the promoter region?

A

After RNA Polymerase II leaves the promoter region, initiation factors dissociate, and the newly synthesized RNA exits the polymerase.

43
Q

How does nucleosome positioning and chromatin remodeling influence RNA Polymerase II transcription?

A

Nucleosome positioning and chromatin remodeling proteins organize large loops of chromatin, helping regulate transcription by allowing or preventing RNA Polymerase II access to DNA

44
Q

What happens to RNA Polymerase II’s CTD phosphorylation status during transcription termination?

A

The CTD becomes dephosphorylated as transcription approaches the end of a gene, which helps in releasing the RNA transcript and resolving the RNA-DNA hybrid.

45
Q

How does ATP-dependent chromatin remodeling facilitate transcription initiation in eukaryotes?

A

ATP-dependent chromatin remodeling proteins move nucleosomes away from promoter regions, allowing transcription factors and RNA Polymerase II to access DNA for transcription initiation.

46
Q

Why is the structure and flexibility of the CTD important for RNA Polymerase II function?

A

The CTD’s flexible structure allows it to interact with various transcription factors and regulatory proteins, and its phosphorylation state provides a platform for regulating different stages of transcription.

47
Q

What is the role of the 5’ cap in mRNA processing?

A

The 5’ cap stabilizes the RNA transcript, protects it from degradation by nucleases, and helps with ribosome alignment during translation.

48
Q

When does 5′ capping of mRNA occur in eukaryotic cells?

A

5′ capping occurs concurrently with transcription as the nascent RNA emerges from RNA Polymerase II, with the CAP-synthesizing complex associating with the CTD.

49
Q

Describe the steps involved in the addition of the 5′ cap to mRNA.

A

The process starts with the removal of one phosphate from the nascent RNA, followed by the addition of guanine in a 5′-5′ triphosphate linkage. The guanine is then methylated on N7, and the first two bases are methylated at the 2′ OH.

50
Q

Why is the 5’-5’ triphosphate linkage in the 5’ cap unusual?

A

It is atypical because guanine is added to the RNA at the 5′ phosphate end, rather than the usual 3′ OH, creating a 5’-5’ triphosphate linkage.

51
Q

What is the purpose of methylation at the 2′ hydroxyl of the first two nucleotides in the 5′ cap?

A

Methylation at the 2′ hydroxyl reduces the reactivity of the RNA, further stabilizing the transcript and protecting it from degradation.

52
Q

How is polyadenylation coupled to transcription termination in eukaryotic mRNA processing?

A

Polyadenylation occurs at the 3′ end of the mRNA during transcription termination, with cleavage of the nascent RNA followed by the addition of a long stretch of adenine residues by the polyadenylation complex.

53
Q

What are the consensus recognition sites required for polyadenylation?

A

Two consensus recognition sites are required for polyadenylation, which bind cleavage recognition factors to cleave the nascent RNA downstream of the gene.

54
Q

What is the purpose of the poly(A) tail in eukaryotic mRNA?

A

The poly(A) tail stabilizes the RNA transcript, aids in nuclear export, and is involved in translation initiation.

55
Q

What is splicing and why is it important in mRNA processing?

A

Splicing is the removal of introns (non-coding sequences) from the pre-mRNA and the ligation of exons (coding sequences). It ensures that only the necessary sequences are present for protein synthesis.

56
Q

How was the discovery of splicing made?

A

Splicing was discovered through hybridization experiments where mature RNA from the cytoplasm was combined with DNA. Electron microscopy revealed loops of single-stranded DNA, indicating that parts of the RNA had been spliced out.

57
Q

How is the 5′ cap of mRNA added during transcription?

A

As the nascent RNA emerges from RNA Polymerase II, a guanine is added in a 5′-5′ linkage to the first nucleotide, which is then methylated at the N-7 position. The first two nucleotides are often methylated at their 2′ OH as well.

58
Q

What is the relationship between the poly(A) tail and transcription termination?

A

The poly(A) tail is added after cleavage of the RNA transcript at the 3′ end during transcription termination by a specialized polyadenylation complex.

59
Q

How does splicing affect the variety of gene products?

A

Splicing allows for different combinations of exons to be joined together, increasing the number of possible gene products from a single gene region.

60
Q

What role does the phosphorylated CTD of RNA Polymerase II play in RNA processing?

A

The phosphorylated CTD coordinates mRNA processing by organizing protein complexes involved in capping, polyadenylation, and splicing during transcription.

61
Q

What was revealed by electron microscopy in the study of splicing?

A

Electron microscopy showed loops of single-stranded DNA that corresponded to intron sequences, indicating that these sequences were spliced out of the mature RNA.

62
Q

What are the four classes of introns based on their splicing mechanisms?

A

The four classes of introns are group I and II self-splicing introns, spliceosome-dependent introns, and protein-catalyzed introns removed by enzymes.

63
Q

How do group II introns undergo splicing?

A

Group II introns undergo splicing via a two-step mechanism where the 2’-OH of an A residue within the intron makes a nucleophilic attack on the 5’ splice site, forming a lariat structure. The 3’-OH of the upstream exon then attacks the 3’ splice site, resulting in exon ligation and intron removal.

64
Q

What is the intermediate structure formed during group II intron splicing?

A

A lariat structure is formed as an intermediate during group II intron splicing.

65
Q

What is the function of small nuclear ribonucleoproteins (snRNPs) in the spliceosome?

A

snRNPs, composed of small nuclear RNAs (snRNAs) and proteins, are essential for recognizing splice sites and catalyzing the splicing reactions within the spliceosome.

66
Q

Which snRNPs are involved in the spliceosome, and what are their roles?

A

The main snRNPs involved in the spliceosome are U1, U2, U4, U5, and U6. U1 recognizes the 5’ splice site, U2 binds to the branch point, and U4/U5/U6 form the tri-snRNP complex for splicing activity.

67
Q

How is ATP used in spliceosome assembly?

A

ATP is required for spliceosome assembly and the activity of helicases that ensure proper RNA pairing and unwinding during splicing.

68
Q

What is the significance of the branch point (BP) in splicing?

A

The branch point (BP) contains a critical adenine residue, which initiates splicing by performing a nucleophilic attack on the 5’ splice site, creating a lariat intermediate.

69
Q

How do U1 and U2 snRNPs contribute to the splicing process?

A

U1 snRNP binds to the 5′ splice site, while U2 snRNP binds to the branch point (BP) and helps position the A residue that initiates splicing.

70
Q

What is alternative splicing, and why is it important?

A

Alternative splicing allows for different exons to be included or excluded from the final mRNA transcript, enabling a single gene to produce multiple protein isoforms with different functions.

71
Q

What percentage of human genes undergo alternative splicing?

A

Over 95% of human genes undergo alternative splicing, leading to the production of multiple protein isoforms from a single gene.

72
Q

What is the impact of alternative polyadenylation on gene expression?

A

Alternative polyadenylation generates different mRNA variants by altering the location of the poly(A) tail, thus affecting mRNA stability, translation, and protein diversity.

73
Q

How does the spliceosome mediate the splicing of introns?

A

he spliceosome mediates intron removal by binding to specific splice sites on pre-mRNA, catalyzing the nucleophilic reactions that join exons and excise introns in the form of a lariat structure.

74
Q

What is the relationship between splicing and RNA-binding proteins?

A

RNA-binding proteins influence splicing decisions by promoting specific splicing pathways, leading to the production of alternative mRNA transcripts.

75
Q

How does splicing contribute to protein diversity in eukaryotic cells?

A

Splicing, particularly alternative splicing, enables one gene to produce multiple mRNA variants, leading to a variety of protein isoforms with distinct functions.

76
Q

Describe the splicing mechanism of group II introns and its similarity to spliceosome-mediated splicing.

A

Group II introns self-splice by forming a lariat structure through nucleophilic attacks by the 2’-OH of an A residue, similar to spliceosome-mediated splicing, which also involves a lariat intermediate and catalysis by snRNPs.

77
Q

What is the role of helicases in the splicing process?

A

Helicases, powered by ATP, are essential for unwinding RNA and ensuring correct snRNP-RNA interactions during spliceosome assembly and function.

78
Q

How does alternative splicing of the calcitonin gene in rats demonstrate protein diversity?

A

Alternative splicing and poly(A) site choice in the calcitonin gene result in the production of different proteins from the same gene, illustrating how splicing contributes to the functional diversity of proteins.

BIOC 302: Molecular Biology (10 decks)

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