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Genetics > Lecture 4: Transcription & Translation > Flashcards

Lecture 4: Transcription & Translation Flashcards

1
Q

What are the basic requirements of transcription?

A

• Ribo-nucleoside triphosphates (NTPs) to create the new strand

• A template to copy

• An enzyme to do the polymerisation: RNA Polymerase

• An energy source

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2
Q

What are two common toxic inhibitors of transcription and translation? How do they work?

A

Alpha-aminitin and Ricin.

• Alpha-aminitin – Amatoxin that inhibits RNA polymerase II, causing protein deficit and ultimately cell death.

• Ricin – Lectin (protein that binds to carbohydrates) is a poison found naturally from castor beans. A-chain inactivates ribosomes by hydrolysing the N-glycosidic bond of an adenosine residue; this inhibits protein synthesis by blocking the binding of elongation factors.

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3
Q

What are some helpful transcription and translation inhibitors? What do they do?

A

Rifampicin and Chloramphenicol.

• Rifampicin – Produces bactericidal antimicrobial activity by inhibiting DNA-dependent RNA polymerase (RNA-P) either by sterically blocking the path of the elongating RNA at the 5’ end or by decreasing the affinity of the RNAP for short RNA transcripts.

• Chloramphenicol – Antibiotic that works by inhibiting protein synthesis by binding to the 50S ribosomal subunit and directly preventing the formation of bacterial protein. Blocks prokaryotic translations.

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4
Q

What are the challenges in transcription?

A

Since not all of a genome is transcribed, the transcriptional machinery must find the correct regions of the genome to transcribe.

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5
Q

How is the challenge of DNA transcription resolved?

A

A gene contains information that directs the transcriptional machinery to the correct place to start and stop transcription. —>
–> Promoter and termination regions.

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6
Q

What is RNA Polymerase (RNAP)?

A

RNA Polymerase is a multi-subunit (multi protein components) enzyme that generates RNA strands (transcripts) using a DNA template and ribonucleotides.

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7
Q

Features and purpose of DNA promoter region.

A

Contains specific sequences to recruit RNA polymerase and regulatory proteins (activators/repressors).

Promoters contain DNA sequences that are recognised by RNA polymerase.

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8
Q

What are RNA transcripts?

A

RNA strands that are synthesised using RNAP.

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9
Q

Describe the transcription termination region.

A

It contains specific sequences to indicate where transcription should stop.

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10
Q

Characteristic of most prokaryotic promoters.

A

They contain conserved consensus sequences at -10 and -35.

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11
Q

What are conserved consensus sequences?

A

By consensus sequences, short stretches of nucleotides that occur multiple times in conserved sequences.

~ Calculated sequence of most frequent residues (nucleotide or amino acid) found at each position in a sequence alignment.

~A sequence of DNA having similar structure and function in different organisms.

By conserved sequences, base sequences in a DNA molecule that have remained essentially unchanged throughout evolution.

Common nucleotides at each position that were preserved through the evolution in species (?)

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12
Q

How does RNAP bind to C.C. sequences in prokaryotic promoters?

A

RNAP binds to these sequences via the sigma subunit (factor).

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13
Q

What is the function of sigma subunit?

A

It conveys promoter specificity to RNA polymerase; that is, it is responsible for telling RNA polymerase where to bind.

Because there are number of different sigma subunits that bind to different promoters and therefore assist in turning genes on and off as conditions change.

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14
Q

What is -35 region responsible for?

A

Rate of transcription.

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15
Q

What is -10 region also known as? What is its function?

A

-10 region is also known as Pribnow box, its role is initiation of transcription.

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16
Q

Where is the first RNA nucleotide transcribed?

A

In the +1 Transcriptional start site.

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17
Q

AT-rich nature of promoter sequences aids what?

A

Separation of the DNA strands.

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18
Q

In what direction does the RNAP move along the template strand?

A

3’ to 5’ direction.

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19
Q

In what direction does the transcription bubble go in?

A

3’ to 5’.

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20
Q

What is transcription bubble?

A

It is formed when the RNAP enzyme binds to a promoter and causes two DNA strands to detach.

It presents a region of unpaired DNA, where a short stretch of nucleotides are exposed on each strand of the double helix.

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21
Q

Non-template strand is also called what?

A

Partner or Coding strand.

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22
Q

Why is the non-template strand called coding strand?

A

Because its sequence will be the same as that of the new RNA molecule, that would end up coding for proteins.

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23
Q

Characteristics of the mechanism of transcription.

A

• Relies on complementary base-pairing.

• Nucleotides are added to the 3’ end of the RNA chain.

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24
Q

How does the elongation process occur in transcription?

A

Addition of ribonucleotides to the 3’ end of the growing script. Nucleophilic attack of -OH.

Hydrolysis of the pyrophosphate releases energy to drive transcription.

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25
Q

Transcription forms a hairpin structure. What does it do?

A

The structure helps slow transcription down.

26
Q

What facilitates transcriptional termination?

A

Hairpin structure of RNA and A-U rich region causes RNA polymerase to pause and release.

A-U base pairs have weak hydrogen bonding.

27
Q

How are eukaryotic mRNAs modified?

A

By adding 7’-methylguanosine to the 5’ end. Known as 5’ cap. (START).

By adding ~250 adenosine nucleotides. Known as the polyA tail. (STOP).

28
Q

What is gene splicing?

A

It happens at the end of transcription process, in which non-coding genes (introns) are cut out and removed.

29
Q

What is ORF Open Reading Frame)?

A

Region to be translated. Between untranslated regions (start and stop).

30
Q

What is an operon?

A

An mRNA structure that is found in prokaryotes that codes for multiple proteins.

31
Q

Why does eukaryotic mRNA need to be modified?

A

To protect the mature mRNA from degradation, and help export it from the nucleus. This gives it stability.

• 5’ cap – modified guanine. Critical for mRNA recognition and proper attachment to the ribosome. Also, protects from nucleases.

• 3’ poly-A tail – promotes export from nucleus. Protects the mRNA transcript from degradation.

• Both cap and tail regulate mRNA splicing, transport, translation, and stability.

32
Q

What is the difference between an operon and ORF (open reading frame)?

A

An open reading frame is the region on a mRNA that is translated by the ribosome i.e it starts with a start codon, has a series of amino acid-coding codons and ends with a stop codon.

An operon contains several linked open-reading frames on a single mRNA i.e this is an RNA that can code for multiple proteins.

33
Q

How many proteins does the 4 letter code of RNA translate into?

A

20 amino acid code of proteins.

34
Q

How is mRNA code read?

A

As a series of triplet codons with each codon specifying either an amino acid or a STOP.

35
Q

What does translation involve?

A

Ribosomes and transfer RNA (tRNA) molecules.

36
Q

What is the start codon?

A

AUG is the start codon, for the vast majority of genes.

37
Q

What is a triplet unambiguous code?

A

A codon that specifies for one amino acid.

38
Q

What is a codon?

A

A group of 3 ribonucleotides.

39
Q

What does it mean when a codon is informative?

A

That it codes for something. Of the 64 possible codons, 61 code for an amino and 3 for a stop codon. There aren’t any ‘blank’ codons.

40
Q

What is a degenerate code?

A

More than one code can make a single acid.

Most amino acids have multiple codons. Often the 3rd base is not important.

41
Q

What are the three stop codons that signify the end of translation?

A

UAA, UAG, UGA

42
Q

What does it mean when referred to tRNA as ‘charged’?

A

When an amino acid is attached to it.

43
Q

How is the genetic code read?

A

Via transfer RNAs (tRNAs)

• Amino acids are attached to specific tRNAs.

• anticodon of the ‘charged’ tRNA base pairs with a complementary codon on the mRNA.

~ Anticodon matches up with the instructions on the mRNA, thus making sure that the right amino acid is incorporated.

44
Q

What is another name for charged tRNAs?

A

Amino-acyl tRNAs

45
Q

How do tRNAs get ‘charged’?

A

Aminoacyl-tRNA synthetase enzymes attach amino acids to specific tRNAs.

There’s a different synthetase enzyme for each amino acid, one that recognises only that amino acid and its tRNAs.

46
Q

What are the characteristics of mechanism of translation?

A

• Charged tRNAs form the link between mRNA and protein.

• Sits of protein synthesis is the ribosome.

• Composed of RNAs + proteins.

• Contains a large and a small subunit.

• Ribosomal RNAs provide the catalytic function for translation.

(RNAs are one of the key components)

47
Q

What are the key sites in ribosomes?

A

E, P & A

Aminoacyl site

Peptidyl site

Exit site

48
Q

What direction does the ribosome move in?

A

5’ to 3’ direction.

49
Q

What are the categories of mutations?

A

• Missense mutation: A mutation that results in an amino acid change but does not change the reading frame of the protein.

• Silent mutation: A mutation that does not result in a change in amino acid.

• Frameshift mutation: A mutation involving insertion or deletion of bases that is not a multiple of three. This shifts the reading frame of all codons following the site of mutation.

• Nonsense mutation: A mutation that changes a codon specifying an amino acid into a stop/termination codon.

50
Q

How does translation get initiated in prokaryotes?

A

• A sequence in the 5’ untranslated region, the Shine-Dalgarno, pairs with the 16S rRNA and places the AUG in the P site of the ribosome.

• A modified version of methionine, N-formylmethionine, is used for initiating amino acid.

51
Q

Why does methionine need to be modified in prokaryotes?

A

It helps them identify and remove methionine residues at the start of proteins.
Removal of the translation initiator N-formyl-methionine or methionine from a recombinant protein is often critical for its function and stability.

Because N-Formylmethionine is present in proteins made by prokaryotes but not in those made by eukaryotes, the immune system can use it to help distinguish self from non-self.

Polymorphonuclear cells can bind proteins starting with N-Formylmethionine, and use them to initiate phagocytosis.

52
Q

How does methionine, N-formylmethionine, initiate amino acids?

A

Methionine is specified by the codon AUG, which is also known as the start codon.
Consequently, methionine is the first amino acid to dock in the ribosome during the synthesis of proteins.

53
Q

How does translation get initiated in eukaryotes?

A

The small ribosomal subunit and initiating tRNAi-Met binds to the 5’ cap and scans the mRNA until the first AUG is reached.

54
Q

What are the eukaryotic ribosomal subunits?

A

60S (5S, 5.8S, 28S RNA) and 40S (18S RNA) = 80S

55
Q

What are the prokaryotic subunits of the ribosome?

A

50S (5S and 23S RNA) and 30S (16S RNA) = 70S

56
Q

State the steps of translation elongation.

A

Initiation, Elongation, and Termination.

• A ‘charged’ aminoacyl tRNA enters the A site pairing codon with anticodon. Elongation factors act as transporters to bring in the aminoacyl tRNA.

• At the same time as peptide bond formation, the covalent bond between the amino acid and tRNA in the P-site is broken.

• mRNA shifts (translocation) by 3 bases. The tRNA that was in the P site now exits via the E site (3’ to 5’). The tRNA with growing polypeptide chain is in the P site and the A site is free.

57
Q

What are the translation elongation factors?

A

Translational elongation factors are proteins that play two important roles during the elongation cycle of protein biosynthesis on the ribosome.

First, elongation factors are involved in bringing aminoacyl-transfer RNA to the ribosome during protein synthesis.

Second, an elongation factor is involved in translocation, the step in elongation at which the peptidyl-tRNA is moved from one ribosomal site to another as the messenger RNA moves through the ribosome.

58
Q

What helps peptide bond formation?

A

Enzyme called peptidyl-transferase.

(The peptide bond formation is catalysed by the 23S rRNA in prokaryotes).

59
Q

What is the purpose of 40S subunit in the eukaryotic ribosome?

A

It decodes the genetic message.

60
Q

What is the function of 60S ribosomal subunit in eukaryotes?

A

It catalyses peptide bond formation.

61
Q

How is translation terminated? State the process.

A

• A stop codon enters the A site.

• Release factors (RF) stimulate hydrolysis of the polypeptide from the tRNA.

• Polypeptide is released, ribosomal subunits dissociate and mRNA and tRNA are released.

Genetics (12 decks)

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