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Molecular Biology > Transcription > Flashcards

Transcription Flashcards

1
Q

Transcription

A

Process where RNA is synthesized using the “template strand” of the DNA

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2
Q

List the 4 steps in the flow of information in a eukaryotic cell

A

A gene is first transcribed to pre-mRNA in nucleus, using the complementary DNA strand as template.

mRNA use is to allow the cell to separate information storage and information utilization.

mRNA use is to greatly amplify the synthetic output of information. (1 DNA template can serve as a template for many mRNA molecules)

mRNA are mobile units that are processed after they which they can travel out of the nucleus into the cytoplasm.

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3
Q

Structure of a double stranded DNA

A

2 bars bottom and top 5’ to 3’ and 3’ to 5’

Each bar consists of non template strand, regulatory sequence, promoter, RNA-coding region inclusive of the transcribed sequence and terminator from left to right.

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4
Q

Promoter

A

Signals the beginning of transcription

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5
Q

Terminator

A

Signals the end of transcription

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6
Q

Regulatory sequence

A

Site for the binding of regulatory proteins.

It is before the promotor

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7
Q

Regulatory proteins

A

Influence the rate of transcription

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8
Q

RNA polymerase function

A

RNA polymerase incorporates nucleotides one at a time (5’ to 3’) to form a strand that is complementary to the DNA template

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9
Q

The 1st step of mRNA synthesis

A

Association of the RNA polymerase to the DNA template at the promoter region, thus initiating the transcription process.

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10
Q

The 3 steps of transcription in prokaryotes

A
  1. Initiation
  2. Elongation
  3. Termination
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11
Q

Initiation step

A

Promoter function as a recognition site for sigma factor.
RNA polymerase is bound to sigma factor, causing it to bind to the promoter.

DNA is unwound to form an open complex following binding of mRNA.

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12
Q

Elongation

A

When 10-12 nucleotides have been successfully incorporated, the sigma factor dissociates from the transcription elongation complex.

The core-enzyme catalyzes the addition of ribonucleotides complementary to the DNA template and joins the ribonucleotides using phosphodiester bonds.

RNA polymerase slides along the template strand in the 3’ to 5’ direction, while it synthesizes RNA in the reverse direction, 5’ to 3’.

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13
Q

Termination

A

When RNA reach the terminator, the terminator and RNA transcript dissociate from the DNA>

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14
Q

RNA transcript

A

Note that the RNA is a copy of a coding region between the promotor and terminator

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15
Q

E.coli RNA polymerase

A

Consists of 5 subunits (beta, beta prime, alpha 1, alpha 2 and omega), which form the core enzyme

There is loose association between DNA and core enzyme, hence RNA chains that are begun are not initiated at proper sites.

Core enzyme + sigma factor become complete RNA polymerase.

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16
Q

Core enzyme and sigma factor

A 
When the σ-factor is bound to
the core enzyme, the enzyme
has strong affinity for
promoter sites on the DNA
strand.

Sigma factor participates in recognition of promotor region.

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17
Q

Promoter binding site

A

The promoter consists of -35 and -10 regions. and that is where the RNA Polymerase binds to.

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18
Q

Closed promoter complex

A

Sigma unit and the other subunits together with the promoter sequence forms the closed promoter complex.

Sigma factor + core enzyme + promoter sequence

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19
Q

Consensus sequences

A

They are conserved and apparent in the transcriptional

promoter region for prokaryotes

20
Q

Termination: Intrinsic termination

A

RNA polymerase recognize the terminator that indicates the end of a prokaryotic gene

Regions of RNA molecules can form secondary structures or hairpin loops that cause transcribing RNA polymerase to stall

RNA transcript then separates from the DNA template and RNA polymerase dissociates

21
Q

What do most terminators code for

A
  • > a single stranded region that folds on itself due to hydrogen bonding between complementary base pairs
  • > the final region containing several uracils (poly-U)
22
Q

2 classes of prokaryotic termination

A

Rho-dependent

Rho-independent (intrinsic termination)

23
Q

Rho protein independent termination

A

Physical modified RNA structure terminates transcription

Poly A tails signal the termination site

RNA molecule getting synthesized have a couple of GC rich regions.

GC rich region fuse with each other and form a hairpin loop.

The poly U is the weak point, allowing the RNA transcript to be removed.

24
Q

Rho protein dependent termination

A

Helicase protein Rho terminates transcription

25
Q

A rho independent terminator

A

Contains an inverted repeat followed by a string of approximately 6 A bases

The inverted repeats are transcribed into RNA

The inverted repeats in RNA fold into a hairpin loop, causing RNA polymerase to pause.

Hydrogen bonds in the AU of the poly U tail break

RNA transcript separates from template, terminating transcription.

Conclusion : Transcription terminates when inverted repeats form a hairpin followed by a string of U bases.

26
Q

Rho dependent termination

A
  • Some terminators require terminator proteins such as the Rho termination factor to be active.
  • Rho dependent terminators lack a poly-U region in the 3’ end of the terminator.
  • A hairpin loop is likely to be present after Rho factor
    binds to a terminator pause site on the RNA molecule which causes the transcribing RNA polymerase to stall.
  • Rho then interacts with the RNA polymerase stalled at the terminator site and catalyzes the dissociation of RNA polymerase from the DNA strand. It then dissociates as well.
27
Q

-35 and -10

A

-35 and -10 upstream from the initiation site.

28
Q

Ribo-nucleoside triphosphate

A

Used to synthesize RNA bases.

Examples are ATP, UTP, GTP, CTP

29
Q

Basic Elements of a Promoter Region in E. coli

A

-> The new RNA chain is transcribed from DNA template and +1 denotes the start of a gene (initiation site).

-> RNA polymerase reads the DNA template in a 3’ to 5’
direction, giving rise to a mRNA strand synthesized from 5’ to 3’.

30
Q

List the 3 types of RNA polymerase

A

RNA polymerase 1
RNA polymerase 2
RNA polymerase 3

31
Q

RNA polymerase 1

A

Transcribes genes encoding large precursor
rRNA, which is processed into 5.8S, 18S and
28S rRNAs

Transcribes genes that code for most of the
ribosomal RNA’s

Note: Large precursor rRNA and small ribosomal RNA

32
Q

RNA polymerase 2

A

Transcribes all protein coding genes, it is key
in transcribing mRNAs

Note: mRNA

33
Q

RNA polymerase 3

A

Transcribes tRNA, 5 SrRNA and other small
rRNAs genes

Note: tRNA and small rRNA

34
Q

Eukaryotic gene structure

A
  • 80 CAAT box
  • 30 TATA box

Start codon to stop codon forms hnRNA (heterogenous nuclear RNA) transcript

AAUAA poly A signal

35
Q

What are the differences between eukaryotes and prokaryotes transcription

A
  1. Transcription in nuclear membrane in eukaryotes , mRNA must leave nucleus
  2. Upstream promoter consists of TATA box at -30 and CAAT box -80 possibly present as well.
  3. Transcription factors facilitate binding of RNA polymerase 2 to the promoter
  4. Enhancers may be present upstream, downstream or inside the gene to enhance transcription
  5. PTM can occur
36
Q

PTM post translational modification

A

From pre-mRNA transcribed, junk DNA is removed before mRNA is formed, pre-mRNA is called heterogenous nuclear RNA (hnRNA)

Undergoes PTM to become mature transcript ready for translation

Includes adding 7mG caps, adding poly A tails and removal of introns

37
Q

Why do we need PTM

A

hnRNA must undergo 3 basic PTM before it is ready to go to cytoplasm

1) Addition of 7mG cap to 5’ end
2) Addition of poly A tail to 3’ end
3) Splicing

38
Q

Addition of 5’ 7mG cap

A
  • > Aids in the export of mRNA from nucleus to cytoplasm
  • > Recognized by cap-binding proteins that enable mRNA to bind to ribosome
  • > Protects 5’ end of mRNA from attack by 5’ ribonucleases
39
Q

Addition of 3’ poly A tail

A
  • Protects 3’ end of mRNA from degradation by 3’ ribonuclease
  • Increases the lifespan of the mRNA
40
Q

Splicing - removal of introns

A
  • Provides contiguous coding sequences for translation by ribosome
  • Plays a role in gene expression via alternate splicing
41
Q

3’ Poly A tail 2 characteristics

A
  • added enzymatically to the 3’ end

- a string of 100-200 adenine nucleotide added after transcription

42
Q

Spliceosome mediated splicing

A
  • Introns within the pre-mRNA contain conserved regions required for splicing.
  • The splicing reaction is catalyzed by a spliceosome consisting of small nuclear ribonucleoproteins (snRNPs).
  • The GU dinucleotide at the 5’ end of intron and the AG dinucleotide at the 3’ end of intron determines the intron boundaries to be spliced
43
Q

How spliceosome mediated splicing work

A
  1. The snRNPs (designated U1, U2, U4, U5 and U6) are made up of small nuclear RNA (snRNA) and proteins.
  2. U1 attaches to the 5’ end of the intron by base-pairing while U2 attaches to an A nucleotide at the branch point.
  3. Others snRNPs (U4, U5 and U6) will then attach to the pre-mRNA to form the spliceosome.
  4. U1 and U4 dissociate from the pre-mRNA while U2 attaches to U6.
  5. A loop (or lariat) is formed which gets excised and the exons are ligated together.
44
Q

Video of spliceosome

A

Snurps are made of small RNA and proteins

1 type of Snurp bind to 5’ end of the intron and another type of Snurp bind to the 3’ end of the intron.

Additional Snurps interact with the complex, bringing the 2 end of the introns together, forming a loop. These gather the snurps and form a spliceosome.

Introns is then cut out, exons are joined or spliced together.

Spliceosome then dissociates as the Snurps are released.

45
Q

Introns

A

Intervening sequences

46
Q

Exons

A

Expressed sequences

47
Q

Lifespan of mRNA in eukaryotes and prokaryotes

A

Different mRNAs within the same cell have distinct stabilities.

  • In bacterial cells, individual mRNAs can survive from seconds to more than an hour, shorter lifespan.
  • In eukaryotes (e.g. mammalian cells), mRNA lifespans range from several minutes to days, longer lifespan.
  • The longer the lifespan of an mRNA the more protein may be produced from that mRNA.

Molecular Biology (9 decks)

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