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1
Q

What are the basic principles of transcription and translation?

A
  • RNA is the bridge between genes and the proteins for which they code
  • Transcription is the synthesis of RNA index the direction of DNA
  • Transcription produces mRNA
  • Translation is the synthesis of a polypeptide, using information in the mRNA
  • ribosomes are the sites of translation
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2
Q

How can transcription initiation differentiate between prokaryotes and eukaryotes?

A

In prokaryotes, translation of mRNA can begin before transcription has finished

  • In a eukaryotic cell, the nuclear envelope separates transcription from translation
  • eukaryotic RNA transcripts are modified through RNA processing to yield finished mRNA
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3
Q

What is a primary transcription?

A

A primary transcription is the initial RNA transcript from any gene prior to processing

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4
Q

What happens to the Thymines if the RNA transcript? Which strand is complementary to the RNA transcript?

A
  • The sense strand of DNA has the same sequence as the mRNA; however in mRNA all of the T’s are replaced with U’s
  • The antisense strand of DNA is complementary to mRNA and is used as the template for mRNA synthesis
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5
Q

What are the 3 regions of a prokaryotic gene?

A
  1. Promoter
  2. RNA coding sequence
  3. Terminators
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6
Q

What is the promoter of the prokaryotic region?

A

Promoter: located upstream of the RNA coding sequence, and ensures the proper location of transcription initiation

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7
Q

What is the RNA coding sequence of a prokaryotic gene?

A

The DNA sequence that is transcribed into RNA

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8
Q

What is the terminator of a prokaryotic gene?

A

Terminator: a sequence downstream of the RNA coding sequence and specifies where transcription will stop

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9
Q

What is the first step in gene expression?

A

transcription

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10
Q

What catalyzed RNA synthesis ?

A

RNA synthesis is catalyzed by RNA polymerase, which pries the DNA strands apart and hooks together the RNA nucleotides

The RNA is complementary to the DNA template strand

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11
Q

What are the 3 stages of transcription?

A
  • initiation.
  • elongation;
  • termination
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12
Q

Describe the initiation of transcription

A
  • during transcription, only one strand of DNA is required as a template
  • there is evidence that genes can be transcribed from any strand.
  • The strand serving as the template for transcription is called the antisense strand and the other strand is called the coding strand or sense strand (same sequences as the RNA)
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13
Q

Where is the prokaryote promoter sequence?

A

The prokaryotic promoter sequences are generally found at -35 and -10 bp from the transcription start site

  • 35 consensus sequence: 5’ -TTGACA-3’
  • 10 consensus sequence : 5’ TATAAT-3’ (also called the pribnow box)

Variation with these sequences results in variation in the binding ability of RNA polymerase and can affect the rate of transcription

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14
Q

How will the coding strand be different from the RNA transcript?

A

Note that the coding strand and the RNA transcript will have the same bases from 5’ to 3’

-With the consideration that uracil is substituted for thymine

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15
Q

How can transcription be terminated?

A

Termination occurs at the termination site of the DNA

-RNA polymerase and the new mRNA transcript is released

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16
Q

What is the function of RNA polymerase?

A

RNA polymerase catalyzes the synthesis of an RNA molecule in 5’ to 3’ direction along the 3’ to 5’ template strand of the DNA

Ribonucleoside triphosphate are the RNA precursor molecules used for RNA synthesis

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17
Q

Compare RNA and DNA synthesis

A

RNA synthesis- RNA polymerase, NTPs precursor, no primer needed, uracil pairs with adenine

DNA synthesis- DNA polymerase, dNTP precursor, primer required for initiation, thymine pairs with adenine

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18
Q

What are the four different types of RNA molecules and what their functions are?

A
  1. mRNA- encodes the amino acid sequence of a polypeptide. mRNAs are the transcripts of protein coding genes
  2. tRNA- brings amino acids to the ribosome during the translation process.
  3. rRNA- combines with ribosomal proteins to form the ribosome. mRNA is translated into protein at the ribosome
  4. snRNA- combines with certain proteins and is involved in RNA processing (example mRNA splicing) in eukaryotes
19
Q

In bacteria, there is only a single….

A

RNA polymerase

20
Q

Differentiate the three different RNA polymerases in eukaryotes

A
  1. RNA polymerase I: 5.8s, 18s and 28s rRNA genes
  2. RNA polymerase II: all protein-coding genes (mRNA) and some snRNAs
  3. RNA polymerase III: tRNA genes and some snRNAs
21
Q

What is bacterial RNA polymerase?

A

RNA polymerase core enzyme is a multi-subunit complex that synthesizes RNA using DNA as a template

RNA polymerase moves stepwise along the DNA

RNA polymerase plus sigma factor (holoenzyme) adheres weakly to bacterial DNA and slides along DNA until it reaches a promoter region where it binds tightly and opens up the double helix to expose a short stretch of DNA

22
Q

Where does the RNA polymerase plus sigma factor bind?

A

To the -10 to -35 regions

23
Q

Briefly describe the method of transcription

A
  1. RNA polymerase plus sigma factor binds to the -35 to -10 regions
  2. DNA molecule is unwound by approximately 17 bp in the -10 region
  3. Transcription begins
  4. Once RNA polymerase synthesizes about 10 nucleotides, interactions of sigma factor with the promoter begins to weaken and breaks
  5. The sigma factor is released and elongation of the RNA transcript continues
  6. Elongation continues until the termination signal is encountered. The termination signal is “self complementary”. The tail of the mRNA forms a short double-helix hairpin loop
  7. The RNA hairpin loop causes physical stress on the enzyme complex which destabilizes the polymerase hold on the RNA. RNA is released. Double helix reforms
24
Q

Describe eukaryotic promoters

A

Consist of a collection of conserved short sequence elements located near the transcription start site

25
Q

What are the conserved short sequence elements in eukaryotic promoters?

A
  1. Element: GC box DNA sequence: GGGCGG position= -70 to -200
  2. Element: TATA Box DNA sequence: TATAA position: -20 to -35
  3. Element: CAAT box DNA sequence: CCAAT position: -80
26
Q

What are the proteins required for transcription initiation(transcription factors)?

A
  1. TATA box binding protein
  2. TFIID
  3. TFIIB
  4. TFIIH
  5. TFIIE
27
Q

What is the function of TATA box binding protein?

A
  • a subunit of TFIID

- binds to TATA box of gene promoter

28
Q

What is the function of TFIID?

A

Transcription factor for polymerase II

Causes a distortion in the DNA helix allowing the recruitment of other transcription factors

29
Q

What is the function of TFIIB?

A

Involved in RNA polymerase interactions -start site recognition (before polymerase)

30
Q

What is the function of TFIIH?

A
  • contains a DNA helicase to unwind DNA

- activates RNA polymerase by phosphorylation

31
Q

What is the function of TFIIE?

A

Involved in positioning RNA polymerase

32
Q

What is the most common eukaryote promoter sequence ?

A

This is located at position -25 and contains the consensus sequence: -5’-TATAA-3’ (TATA Box)

33
Q

Explain the functioning of the transcription initiation factors

A

A. TBP(subunit of TFIID)/ TFIID bind to the promoter at the TATA box (-25)

B. TFIIB binds to promoter

C. RNA polymerase, TFIIE, and TFIIH are recruited to the promoter and transcription initiation site

D. TFIIH unwinds the DNA

E. TFIIH phosphorylates RNA polymerase activating it for transcription (C-terminal domain)

Most transcription factors are then released from the basal transcriptional machinery prior to transcription initiation

34
Q

What forms the basal transcriptional machinery ?

A

The RNA pol II and the transcription factors TFIID( TAFs and TBP), and other transcription factors form the basal transcription machinery

35
Q

Is the basal transcriptional machinery sufficient for high levels of transcription?

A

The basal transcriptional machinery is sufficient for only a low level of transcription

-For increased levels of transcription or tissue specific transcription, the binding of an activator and mediator/ adaptor molecule is required

36
Q

Explain the fidelity of RNA polymerase

A
  • RNA polymerase lacks the proof reading activity that is found in DNA polymerase
  • the accuracy of transcription is much less than that 9f replication
  • error rate is about 1/10,000 base pairs
  • this level of error is tolerated in the cell because there are many transcripts from each gene and most will be error free
37
Q

What is Supercoiling?

A

There are approximately 10 nucleotide pairs per helical turn if a double helix

  • If you unwind one helical turn, the double helix will rotate one turn
  • DNA with fixed ends, rotation is prevented
  • When 10 DNA base pairs is unwound, the DNA will form one supercoil to relieve tension
  • One supercoil forms for every 10 bp opened up
  • In this case a positive supercoil
38
Q

What are negative and positive supercoil?

A

When a protein is tracking through the DNA, the ends are unable to rotate

  • Excess helical turns ahead of the protein cause positive supercoil
  • Deficit of helical turns behind the protein cause negative supercoils

Negative Supercoiling- helix opening facilitated

Positive Supercoiling- helix opening hindered

39
Q

What types of supercoils are formed due to transcription?

A

Transcription of DNA results in positive supercoils in front of the RNA polymerase and negative supercoils behind the RNA polymerase

Under wound DNA already transcribed causes negative supercoils. Topoisomerase relaxes negative supercoils

Overwound DNA yet to be transcribed causes positive supercoils. Gyrase introduces negative supercoils

40
Q

How can intrinsic termination sequences be involved in the termination of transcription?

A
  1. Palindromic regions that form hairpins varying in length from 7 to 20 bps
  2. The stem loop structure includes a G-C rich region and is followed by a run of U bases
41
Q

How can extrinsic factors be involved in the termination of transcription?

A

Rho proteins highly important

  1. Rho protein attaches to recognition site on the RNA.
  2. Rho moves along RNA following RNA polymerase
  3. RNA polymerase pauses at terminator and rho catches up
  4. Rho unwinds DNA-RNA hybrid and pull RNA from the RNA polymerase
  5. Termination: RNA polymerase, rho, and RNA are released
42
Q

What is RNA processing?

A

Enzymes in the eukaryotic nucleus modify pre-mRNA (RNA processing) before the genetic messages are dispatched to the cytoplasm

  • DURING RNA processing, both ends of the primary transcript are usually altered
  • Also, usually some interior parts of the molecule are cut out, and the other parts spliced together
43
Q

Explain the functional and evolutionary importance of introns

A
  • Some introns contain sequences that may regulate gene expression
  • Some genes can encode more than one kind of polypeptide, depending on which segments are treated as exons during splicing
  • This is called alternative RNA splicing
  • Consequently, the number of different proteins an organism can produce is much greater than its number of genes

Molecular Biology (24 decks)

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