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Biochem Exam 2 > Transcription > Flashcards

Transcription Flashcards

1
Q

What is the central dogma?

A

DNA –> RNA –> Protein
replication: DNA –> DNA
transcription: DNA –> RNA
translation: RNA –> protein (primary structure)

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2
Q

what is gene expression and its properties?

A

definition: the process by which the information encoded in a DNA sequence is converted into a product (RNA or protein) that has some effect on a cell or organism

a. The first step is transcription
b. Many identical RNA copies can be made from the same gene
c. Each RNA molecule can direct the synthesis/translation of many identical protein molecules → successive amplification that enables cells to rapidly synthesize large amounts of proteins whenever necessary
d. Each gene can be transcribed and its RNA translated at different rates and cells can change/regulate the expression of its genes according to the cell’s needs

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3
Q

How does RNA’s structure affect its function?

A

largely single-stranded but it contains short stretches of nucleotides that can base-pair with complementary sequences or nonconventional base-pair interactions –> can fold up into a variety of shapes just as a polypeptide chain folds up to form the final shape of a protein (DNA cannot do this)

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4
Q

definition of transcription?

A

the process in which the cell copies the nucleotide sequence of that gene into RNA

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5
Q

What is the transcription bubble?

A

the localized region of unwound DNA, creating an open space (aka bubble) that forms during transcription when RNA polymerase binds to the promoter of DNA
- RNA polymerase
- the DNA
- the RNA product
the bubble continuously moves along the DNA as DNA is unwound ahead and then rewound behind while the RNA produce is extruded from the complex until it reaches a terminator sequence

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5
Q

What are the differences between transcription and DNA replication?

A

RNA does NOT remain hydrogen-bonded to the DNA template strand (unlike a newly formed DNA strand)

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6
Q

Difference between RNA polymerase and DNA polymerase?

A

a. RNA polymerase: can start an RNA chain without a primer
b. RNA polymerase backtracks to proofread and lacks exonuclease activity; DNA polymerase does not have backtracking but has more efficient exonuclease proofreading to remove an incorrect nucleotide (error rates: 10^4 for RNA polymerase vs 10^7 for DNA polymerase)

RNA polymerase uses ribonucleoside triphosphates as substrates and catalyzes the linkage of ribonucleotides, NOT deoxyribonucleotides like DNA polymerase does

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7
Q

ribosomal RNAs (rRNAs) function?

A

form the core of the ribosome’s structure and catalyze protein synthesis

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8
Q

mRNAs function?

A

code for proteins

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9
Q

microRNAs (miRNAs)

A

regulate gene expression

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10
Q

transfer RNAs (tRNAs)

A

serve as adaptors between mRNA and amino acids during protein synthesis

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11
Q

RNA polymerase structure and active site

A

Both prokaryotic and eukaryotic RNA polymerase contain a central metal ion (green) in their active sites
RNA polymerases consist of 5 subunits in E. coli and require 2 Mg2+ at the active site
The 3’-hydroxyl group of the growing RNA chain attacks the alpha-phosphoryl group of the incoming nucleoside triphosphate → results in the release of pyrophosphate
RNA synthesis occurs in a complex called the transcription bubble where around 17 bases of the DNA are unwound

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12
Q

What are the functions of RNA polymerases during transcription?

A

a. Search for and bind to promoter sequences (the initiation sites for RNA synthesis)
b. Unwind a short stretch of the double helix DNA to reveal the bases to be transcribed
c. Select the ribonucleoside triphosphate that corresponds to the DNA template and catalyze the formation of a phosphodiester linkage
d. Detect termination signals that halt transcription
e. Interact with other proteins that regulate the process of transcription

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13
Q

What are the stages of RNA synthesis?

A
  1. Initiation: a promoter site in the DNA is identified to guide which part of DNA to be used as the template for RNA synthesis
  2. Elongation: extend/synthesize RNA in the 5’ to 3’ direction
  3. Termination: end transcription
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14
Q

what are the template strand and coding strand?

A

Template/antisense (-) strand: the DNA strand that is used to guide the synthesis of the RNA molecule
Coding/sense (+) strand: the nontemplate DNA strand (its sequence is equivalent to the RNA product)

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15
Q

What number is used to denote the first nucleotide to be transcribed?

A

+1
the nucleotide right before is denoted with -1
(negative = upstream of first nucleotide to be transcribed; positive = downstream)

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16
Q

what is a ribonucleoside triphosphate?

A

a molecule composed of a ribonucleoside (ribose sugar attached to a nitrogenous base) + 3 phosphate groups (ie. ATP, GTP, CTP, and UTP)

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17
Q

what is translocation?

A

the movement of RNA polymerase along the DNA template during the elongation phase of RNA synthesis. After RNA polymerase adds a new nucleotide to the growing RNA strand, it needs to shift (or “translocate”) along the DNA by one base pair to expose the next DNA nucleotide for transcription

as the bubble moves, the RNA product exits the enzyme and the transcribed DNA rejoins its partner

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18
Q

how does elongation work?

A

Occurs when a ribonucleoside triphosphate base-pairs with a nucleotide on the DNA template

The 3’-hydroxyl group of the last nucleotide in the chain attacks the alpha-phosphoryl group of the incoming nucleoside and triphosphate → reforming a phosphodiester linkage and releasing PPi (pyrophosphate)

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19
Q

How does RNA polymerase proofreading work?

A

The RNA-DNA hybrid can move in the direction OPPOSITE that of elongation (backtracking is LESS favorable energetically than moving forward (elongation) since it breaks the bonds between a base pair via hydrolysis)

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20
Q

Why is the lower fidelity (aka higher error rate) of RNA polymerase permitted?

A

because RNA errors are not transmitted to progeny (offspring) the way DNA is

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21
Q

Difference in prokaryotic and eukaryotic RNA polymerases?

A

Prokaryotes: use ONE RNA polymerase for ALL types of RNA transcription
Eukaryotes: use THREE slightly different RNA polymerases → each of the three RNA polymerases is responsible for transcribing a unique type of RNA
RNA polymerase I
RNA polymerase II
RNA polymerase III
similarity: Both contain a central metal ion (green) in their active sites

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22
Q

how many and what are the subunits of prokaryotes RNA polymerase?

A

5 core subunits:
2 alpha subunits
1 beta subunit
1 beta prime subunit
1 omega subunit
not part of core enzyme
1 sigma subunit

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23
Q

what is the holoenzyme of prokaryotes RNA polymerase?

A

the holoenzyme is formed when the core enzyme (α2ββ’ω) associates with a sigma factor (σ). The complete structure is represented as α2ββ’ωσ

The primary role of the holoenzyme is to initiate transcription: the sigma factor enables the RNA polymerase to recognize specific promoter sequences in the DNA –> facilitating the binding of the polymerase to the promoter region
Once transcription is initiated, the sigma factor may be released –> allows the core enzyme to proceed with the elongation phase of RNA synthesis

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24
Q

What is the core enzyme of prokaryotes RNA polymerase?

A

Composed of 5 subunits (α2ββ’ω)
primarily responsible for the actual synthesis of RNA (elongation), but CANNOT initiated transcription without the sigma factor/subunit

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25
Q

What and where are the core promoters of E.coli?

A

the core promotor:
-10 sequence: TATAAT
-35 sequence: TTGACA

promoter sequences may vary though from this. but generally, the more similar a promoter sequence is to the consensus sequence (the most common nucleotides found at each position), the more effective the promoter is

26
Q

How does the asymmetry of the promoter in E.coli affect transcription initiation?

A

the core promoter (-10 and -35 sequences) is orientated in a specific way on the DNA double helix
RNA polymerase binds to the promoter asymmetrically, with its active site oriented towards the template DNA strand –> determines the direction RNA polymerase will move along the DNA

27
Q

How does the sigma factor of prokaryotic RNA polymerase help transcription?

A

The sigma subunit of the holoenzyme is responsible for recognizing the promoter sequence
Once the promoter is located, transcription begins, and when the pre-mRNA is 9 to 10 nucleotides long, sigma factor is released and will go assist another enzyme to find the promoter

28
Q

How many sigma subunits does E.coli have?

A

E. coli has SEVEN distinct sigma subunits that recognize different promoters

29
Q

What are the steps for bacterial transcription?

A
  1. bacterial RNA polymerase RNAp contains a subunit, sigma factor that recognizes the promoter of the gene
  2. once transcription has begun, sigma factor is released and the RNA polymerase moves forward to continue synthesizing the RNA until it reaches a terminator sequence
  3. after transcribing this sequence into RNA, RNAp halts and releases both the DNA template and the newly made RNA transcript
  4. RNAp then associates with a free sigma factor and searches for another promoter to begin the process again

NOTE: The regions transcribed into RNA INCLUDE the TERMINATOR but NOT the PROMOTER nucleotide sequences

30
Q

How does bacterial transcription termination occur?

A

The RNA complement of the DNA stop signal (terminator sequence) forms a HAIRPIN structure that is followed by several URACIL residues
Upon synthesis of the hairpin, the RNA polymerase stalls → causing the RNA product to be released and the DNA double helix to reform

31
Q

What is the simplest stop signal in bacterial transcription?

A

the transcribed product of a segment of palindromic DNA (the sequence of nucleotides reads the same in the 5’ to 3’ direction on one strand as it does in the 5’ to 3’ direction on the complementary strand)
ie. 5’ - GAATTC - 3’
3’ - CTTAAG - 5’

32
Q

differences in eukaryotes and prokaryote DNA?

A

Eukaryote: linear DNA within the nucleus
prokaryote/bacteria: circular DNA; no nucleus or membrane bound organelles

33
Q

what are 3 important characteristics that influence gene expression in eukaryotes?

A
  1. The nuclear membrane that separates the site of RNA synthesis from that of protein synthesis
    RNA synthesis: in nucleus
    Protein synthesis: in ribosomes (cytosol)
  2. Complex transcriptional regulation
  3. RNA processing: pre-mRNA –> MRA
    eukaryotes use differential gene regulation to generate different cell types
34
Q

What are the 3 RNA polymerases eukaryotes use and what type of genes do they each transcribe?

A

RNA polymerase I: transcribes most rRNA genes
RNA polymerase II: mRNAs transcribes ALL protein-coding genes (miRNA genes + genes for other noncoding RNAs → ie. those for the spliceosome)
RNA polymerase III: transcribes tRNA genes, 5S rRNA gene, and genes for many other small RNAs

35
Q

How do the three eukaryotic RNA polymerases differ in terms of alpha-amanita effects and relative abundance (total mass) in cells?

A

RNA polymerase I:
Insensitive to the effects of alpha-amanita
Relative abundance (total mass) in cells: rRNA 98%

RNA polymerase II:
Strongly inhibited by alpha-amanita
Relative abundance (total mass) in cells: mRNA <1%

RNA polymerase III:
Inhibited by high concentrations of alpha-amanita
Relative abundance (total mass) in cells: tRNA 1%

36
Q

How do the 3 eukaryotic RNA polymerases differ?

A

The 3 RNA polymerases differ in DNA substrate specificity, location, and sensitivity to the toxin alpha-amanitin

37
Q

What is the toxin alpha-amanitin?

A

Toxin alpha-amanitin is produced by the poisonous mushroom Amanita phalloides
Alpha-amanitin: primarily inhibits RNA polymerase II → prevents the synthesis of mRNA → stops protein synthesis → leads to cell death

38
Q

How does RNA polymerase II differ in terms of structure compared to the other 2 RNA polymerases?

A

All of the polymerases are similar in structure
BUT RNA polymerase II has a unique domain (carboxyl-terminal domain CTD) that regulates RNA polymerase II activity by phosphorylation mainly on the serine residues of the CTD
- RNA polymerase II must be phosphorylated (phosphorylation of its C-terminal domain) to start transcription

Carboxyl-terminal domain: the phosphorylation of the serine residues in the CTD is tightly regulated and changes at different stages of transcription → serves as a signal to recruit the appropriate proteins needed for the 3 steps of transcription

39
Q

What are cis-acting elements?

A

sequences on the SAME DNA strand as the genes being transcribed (template strand) that regulate and influence the expression of genes

40
Q

What are common elements can be found in the RNA polymerase II promoter region?

A
  1. TATA box: located between positions -30 and -100 base-pairs upstream of the initiation site
  2. The initiator element (Inr): located between base-pairs -3 and +5 and is often paired with the TATA box
  3. The downstream core promoter element (DPE): located between base-pairs +28 and +35; works in cooperation with Inr when the TATA box is absent
  4. Other regulatory elements ie. CAAT box and GC box: located between -40 and -150 base-pairs upstream (GC box is common in genes that are continuously expressed)
41
Q

How do transcription factors regulate the initiation of transcription for eukaryotic cells? Give the key steps

A
  1. TFIID binds to the promoter (ie. TATA-binding protein, a subunit of TFIID binds to the TATA box)
  2. TFIID creates a dramatic local distortion in the helix –> other TFII factors (ie. TFIIA, B, E, F, H) are recruited to the promoter region –> This assembly forms the active transcription complex which helps RNA polymerase II recognize the transcription start site
  3. TFIIH unwinds the DNA and phosphorylates RNA polymerase II to officially initiate transcription
  4. after transcription begins, the general transcription factors dissociate from DNA except for TFIID
42
Q

What is the most commonly recognized cis-acting element for genes transcribed by RNA polymerase II?

A

TATA box

43
Q

enhancers

A

cis-acting elements that have NO promoter activity BUT can stimulate the effectiveness of promoters even when located thousands of nucleotides from the start site of transcription

  • Increases the rate of transcription
  • can be upstream, downstream, or even in the middle of a transcribed gene
  • operate in conjunction with specific enhancer-binding proteins
44
Q

How many rRNAs are produced from one eukaryotic pre-rRNA?

A

RNA polymerase I processes eukaryotic pre-rRNA and produces 3 rRNA

45
Q

What are the three RNA processing steps pre-mRNA undergo to become mature mRNA?

A

5’ end capping
3’ end poly-A tail addition (polyadenylation)
RNA splicing

capping and polyadenylation occur on ALL RNA transcripts and RNA splicing occurs on almost all

46
Q

describe 5’ capping

A

5’ end modified by addition of a 5’ cap where a GTP (a modified guanine nucleotide) is added to the precursor in an unusual 5’-5’ linkage (the cap may also be methylated –> 7-methylguanosine cap)
–> the cap protects the mRNA from degradation by 5’ exonucleases

Capping takes place after RNA polymerase II has produced about 25 nucleotides of RNA
In bacteria: the 5ʹ end of an mRNA is simply the first nucleotide of the transcript

47
Q

describe RNA splicing

A

introns (noncoding stretches of RNA) are removed and the products are ligated to form mature mRNA

48
Q

describe polyadenylation (addition of a poly-A tail)

A

The 3’ end is first cleaved by a specific endonuclease that cuts the RNA chain at a particular sequence of nucleotides → then a 2nd enzyme adds a series of repeated Adenines to the trimmed end (around 250 nucleotides in length)

49
Q

small nuclear ribonucleoproteins (snRNPs)

A

recognize splice-site sequences through complementary base-pairing between their RNA components and the sequences in the pre-mRNA; they carry out the chemistry of splicing
Form the core of the spliceosome

50
Q

Spliceosome

A

the large assembly of RNA and protein molecules (small nuclear ribonucleoproteins snRNPs) that carries out RNA splicing in the nucleus

51
Q

what is the lariat structure?

A

the lasso-shaped loop (‘loop with a tail’) formed through a covalent bond between the 5’ end of the intron and the branch point adenine
the intron in pre-mRNA forms this lariat structure and is excised and then degraded in the nucleus

52
Q

What are the steps of RNA splicing?

A
  1. snRNP U1 recognizes the 5ʹ splice site and U2 recognizes the lariat branch-point site through complementary base-pairing
  2. U6 then “re-checks” the 5ʹ splice site by displacing U1 and base-pairing with this intron sequence itself
  3. Conformational changes in U2 and U6 drive the formation of the spliceosome active site
  4. the spliceosome comes and splices off the introns and then marks the splice site as successfully complete
53
Q

When and where does RNA splicing occur?

A

RNAs processing in the NUCLEUS
Splicing typically begins before an RNA polymerase finishes transcribing a gene
→ A “pre-mRNA transcript” containing all of the gene’s introns and exons rarely exists inside the cell

54
Q

what is alternative splicing and some of the rules?

A

allows many different proteins to be produced from the same gene
–> the reason why eukaryotic genes have intron-exon arrangement

properties/rules:
- All exons are transcribed, BUT they can be skipped over by the spliceosome to produce alternatively spliced mRNAs
- Skipping occurs when the splicing signals at the 5ʹ end of one intron are paired up with the branch-point and 3ʹ end of a different intron
- Exons can be skipped or included, BUT their order is specified in the DNA sequence and can NOT be rearranged

55
Q

operons

A

a group of genes that are transcribed and translated together as a single mRNA molecule and under the control of a SINGLE promoter, operator, and terminator (functionally related genes all working towards a shared end goal)
ONLY in prokaryotic cells

56
Q

difference in how eukaryotic and prokaryotic cells code for tryptophan? (Trp operon)

A

Prokaryotic cells: five genes are involved in making the amino acid tryptophan → trp operon
the operon produces ONE mRNA which is then translated into 5 proteins
- When activated, ALL 5 enzymes are produced simultaneously

Eukaryotic cells: The 5 separate genes are spread over 4 different chromosomes. Each gene produces one mRNA and one protein –> the total is 5 seperate mRNAs produced and each of the different mRNAs produce the different 5 proteins

57
Q

difference between prokaryotes and eukaryotes in transcription and translation?

A

prokaryotes:
- only exons, no introns
- no alternative splicing
- transcription and translation both occur in the cytosol
- mRNA does not undergo any processing (once transcribed it is ready for translation immediately)

eukaryotes:
- includes both exons and introns
- yes alternative splicing
- transcription occurs in the nucleus; translation occurs in the cytosol
- the transcribed RNA transcript is pre-mRNA and needs to undergo RNA processing to become mature mRNA ready for translation

58
Q

differences in DNA replication and transcription?

A

DNA replication:
- RNA primer needed
- DNA polymerase proofreading has exonuclease activity and is lower error rates
- replicates the entire DNA molecule
- DNA replication is faster than transcription on average

transcription (RNA synthesis):
- no primer needed
- RNA polymerase proofreading consists of backtracking which is less favorable energetically than moving forward and has higher error rates
- only portions of DNA sequences are transcribed into RNA
- transcription is slower on average than DNA replication

59
Q

differences between DNA and RNA?

A

DNA:
- predominantly double stranded
- b-form double helix
- deoxyribose sugar (doesn’t have the C2 OH group; more stable)
- has thymine
- self replicating
- mostly found in the nucleus
- exons and introns
- longterm storage of genetic info
- lower error rates (higher fidelity)

RNA
- largely single stranded
- a-form helix
- ribose (C2 has a OH group; less stable)
- has uracil
- RNA is synthesized from DNA using one DNA strand as a template on an as-needed basis
- found throughout the cell
- exons only
- transfers genetic code from nucleus to ribosomes to make proteins
- higher error rates (lower fidelity)

60
Q

Splice sites

A

the specific sites where splicing occurs at (located at the 5’ donor splice site and 3’ acceptor splice site of an intron)

61
Q

alternative splicing rule

A

One splice site should come from one intron at the 5’ end of the GENE, and the other should come from a different intron at the 3’ end of the GENE
–> alternative splicing products produced must include both the first and last exons of the gene

62
Q

Ribosome

A

a large complex made from dozens of small ribosomal proteins and several ribosomal RNAs (rRNAs) that latches onto an mRNA → captures and positions the correct tRNA molecules → then covalently link the amino acids that they carry to form a polypeptide chain
what actually decodes the mRNA message

63
Q

what are the ribosomal sites and the order they go in?

A
  1. A site
  2. P site
  3. E site

Biochem Exam 2 (6 decks)

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