Eukaryotic Transcription and the Genetic Code

Question 1

Which RNA polymerase codes for what?

Answer 1

I : rRNA
II : mRNA
III: rRNA and tRNA

Question 2

What is TBP and what does it do?

Answer 2

TATA box binding protein which is part of a large protein complex called transcription factor II D. TBP consists of two similar domains that bind to DNA and cause it to bend.

Question 3

What are general transcription factors?

Answer 3

The proteins that bind to upstream control elements and recruit further proteins to help bind RNA polymerase II for transcription. These proteins are able to bind to DNA following TBPs recruitment to the TATA box as the bending of the DNA exposes the sequences allowing them to recruit the general transcription factors.

Question 4

How do cis acting proteins have an effect on transcription?

Answer 4

These regulator proteins have an effect on the promoter region. As they they are far from the start site the DNA bends bringing them closer and there effect is acted out through a mediator complex
‘strand’
‘bend’ ‘mediator’
‘strand’

Question 5

How can chromatin structure affect transcription?

Answer 5

If the DNA to be transcribed is wrapped around a nucleosome then the TBP cant bind to the TATA box and therefore the general transcription factors cant either. Chromatin remodeling is required to expose the promoter regions.

Question 6

How are DNA strands separated for transcription in eukaryotes?

Answer 6

Eukaryotic DNA strands are split via the activity of transcription factor II H (a big complex), which requires ATP hydrolysis. This transcription factor also mediates the initaion of transcription

Question 7

How does RNA polymerase II escape the promoter region?

Answer 7

Two kinases that are part of the TFIIH complex phosphorylate amino acids that are part of the C terminal of the same complex. This allows RNA polymerase to disengage and from the general transcription factors and begin to elongate the RNA transcript. After this, all the other transcription factors will dissociate from DNA but most of TFIID will remain bound to the RNA polymerase.

Question 8

Why must eukaryotic RNA be modified before translation and how?

Answer 8

RNA is transcribed in the nucleus and therefore must undergo modifications to keep it stable while it is exported to the cytoplasm and translated. The phosphorylated C terminal of TFIID allows for the binding of enzymes that will facillitate the three necessary modifications which are
- Capping
- Splicing
- Polyadenylation

Question 9

How is the mRNA transcript capped and why?

Answer 9

The 5’ end of the RNA strand is capped in order to protect it from being degraded. Capping: the terminal phosphate group is removed and a guanine is added forming a 5’ - 5’ triphosphate bridge and the guanine is methylated at the N7 position creating 7-methylgaunosine which blocks enzymes from degrading the transcript. Capping can occur anytime after 25 nucleotides have been added.

Question 10

Why are mRNA transcripts spliced?

Answer 10

Pre-mRNA transcripts are ‘spliced’ to remove introns as they do not code for amino acids and would disrupt translation. Splicing can also involve the removal of exons. Splicing can occur during transcription once the 5’ end has been capped. (Because of introns eukaryotic transcription takes much longer than prokaryotic).

Question 11

How does splicing add genetic diversity?

Answer 11

Splicing can include exons or exclude others allowing for different forms of a protein called isoforms to be made as and when they’re needed.

Question 14

How are specific sequences targeted for splicing?

Answer 14

Each intron is bordered by a sequence that indicates the splice site. Each intron starts with a GU on its 5’ side and an AG on the 3’ end. In addition somewhere in the intron there is an A that is part of a branching site, which is important for excision

Question 15

How are introns removed?

Answer 15

The branch site is a bent region of the transcript. The 2’ OH group on the transcript is therefore bought into close proximity to the GU 5’ end border. The OH performs a nucleophilic attach on the G cutting the 5’ end. The newly exposed OH group on the exon then performs a nucleophilic attack on the on the last the last G on the 3’ end of the intron cutting that end. This forms an intron lariat which is removed and the ends of the exons are joined together.

Question 16

Where does splicing occur?

Answer 16

In the spliceosome - a protein complex containing small nuclear RNAs. the spliceosome acts an enzyme for the process.

Question 17

How is transcription terminated?

Answer 17

RNA polymerase will reach a terminator sequence where it will stop transcribing. Before DNA polymerase dissociates it will transcribe the polyadenylation signal which codes the enzymes that will cleave off the mRNA transcript.

Question 18

What does the polyadenylation signal do?

Answer 18

Cleaves the RNA exposing the 3’ polyadenylation site. Poly-A-polymerase (PAP) will then bind to the 3’ end of the transcript and add around 200 adenines without a template. This poly A tail provides stability for the mRNA (for export from the nucleus) and promote translation. mRNAs are the only RNAs with poly A tails.

Question 19

How can only 4 nucleotides code for 20 different amino acids and why are codons only three bases long?

Answer 19

If one amino acid was coded by one base that means there would only be 4 amino acid, 1 amino acid per 2 bases would be 16. But 1 amino acid per 3 bases = 64 amino acids (4^3). This is why a codon is three bases and how multiple codons can code for the same amino acid.

Question 20

What is the most common type of non coding RNA and how do they work?

Answer 20

tRNA’s recognise codons via their anticodons (a complementary sequence to the mRNA) and the amino acids.

Question 21

What is the rough structure of a tRNA?

Answer 21

Cloverleaf: 3x hair pin structures (the leaves) with a double stranded ‘stem’ with a protruding 3’ end that attaches the amino acid. The hairpin directly below the ‘stem’ contains the anticodon sequence that is essentially exactly the same as the DNA sequence the mRNA transcribes from

Question 22

Why would exact tRNA sequences be inefficient what is natures alternative?

Answer 22

If tRNA’s anticodons where matched exactly, 64 tRNAs would be required. Most amino acids can be coded for by multiple different codons, that often have the same first two bases. The third base of the anticodon can “wobble” (the first 2 bases always match) allowing it to bind to a base thats not its normal partner. This is called non-Watson-Crick base pairing.
- G can bind to C or U
-U can bind to A or G
This can can occur because either
- there is extra space at the third nucleotide which allows the bit of RNA to move away from the complementary strand meaning the third base pair doesn’t bind.
- or there can be a 6th nucleotide included: inosine

Question 23

What is inosine?

Answer 23

A form of adenosine that has been modified by a specific tRNA deaminase. It can bind to A, C or U allowing for wobble to occur.

Question 24

How does having amino acids that are coded for by more than one codon help?

Answer 24

It creates a high level of redundancy in the system to account for any issues with tRNA availability

Question 25

What is special about methionine and tryptophan?

Answer 25

Both are only coded for by one codon.
-Methionine is coded for by the start codon and therefore all proteins start with this the first amino acid.
-Tryptophan is the least abundant amino acid.

Question 26

Where does the genetic code vary?

Answer 26

Mitochondria have their own genome within the organelle. This is most likely due to mitochondria evolving from bacteria and eukaryotic symbiosis. Everywhere else the genome is conserved.

Question 27

What are point mutations?

Answer 27

When there is a change in the sequence of a gene either by
-Insertion
-Deletion
-Substitution
of a nucleotide resulting in either missense, nonsense, silence or frameshift mutation. This can occur of multiple nucleotides.

Question 28

What are silent mutations and there causes?

Answer 28

Silent mutations do not result in a change of amino acid and are caused by substitution point mutation. This generally occurs in the third nucleotide of a codon, sometimes the second but never in the first. The mutation is silent because of tRNA wobble, so the same amino acid is coded.

Question 29

What are missense point mutations and their causes?

Answer 29

When a change in nucleotide changes the amino acid that is coded. This most often occurs in the first nucleotide of the codon but can happen in the other positions.
Missense can be considered conservative or non-conservative
-Conservative = mutant amino acid similar in properties
-Non-conservative =mutant amino acid doesn’t have similar properties

Question 30

What are the properties that vary with different amino acids? What does this mean a missense mutation can cause in a protein.

Answer 30

-Charge
-Hydrophilic/Hydrophobic and the extent as such
-Size
-Functional group in general
Non-conservative missense mutations will likely change the structure of protein.

Question 31

What is a nonsense mutation?

Answer 31

These mutations introduce a stop codon in the of the protein. This truncates the protein either severely impeding its ability to function or stopping it all together. Nonsense mutations are considered one of most serious mutations.

Question 32

What are frameshift mutations?

Answer 32

When a nucleotide is inserted or deleted the frame reading of the codons changes. If a nucleotide is deleted and there is still an AUG start codon frameshift mutations can be harmless. If a nucleotide is inserted the result will be a change in the amino acids coded which will drastically change the produced protein often making the nonfunctional. The same negative result can happen if a nucleotide is deleted depending on where it is.

Question 33

How can silent mutations be deleterious?

Answer 33

Not all tRNA’s are equally prevalent and are generally proportional to prevalence of their respective codons. If the mutation changes the codon to one that is rare there might be an issue in the availability of the required tRNA slowing translation and affecting gene expression.

Question 34

What is required for tRNAs to work?

Answer 34

1. Amino acid activation:
   Activation means that the amnio acid is bound to ATP via the enzyme aminoacyl-tRNA synthetase
   This results in an aminoacyl adenylate intermediate.
2. tRNA charging:
   The intermediate then undergoes a nucleophilic attack by an uncharged tRNA to then be joined by an ester bond thus charging the tRNA. Aminoacyl-tRNA synthetase also facilitates this reaction. The tRNA now has an amino acid attached ready for translation.

Question 35

How are amino acids activated?

Answer 35

By binding to ATP to the OH group on an amino acid.

Question 36

What does aminoacyl-tRNA synthetase do?

Answer 36

Facilitates amino acid activation and tRNA charging. There is an aminoacyl-tRNA synthetase for each amino acid, so 20 different ones. Each synthetase recognises the anticodon of the tRNA through complementary binding sites. Each synthetase also has a synthesis site with a specific affinity and is where activation/charging occurs. These synthetases also have an editing function and therefore check and correct if the wrong amino acid has been added to the tRNA. The correct amino acid would be unable to enter the editing site. When an amino acid has been charged by aminoacyl-tRNA synthetase the complex formed is called an aminoacyl adenylate intermediate.

Question 37

How are tRNAs charged?

Answer 37

The aminoacyl adenylate intermediate undergoes a nucleophilic attack by an uncharged tRNA joining the two via an ester -> tRNA is charged/bound to an amin acid. Aminoacyl synthetase is the same enzyme that facilitates tRNA charging and amino acid activation.

Question 38

What is difference between the two subunits of ribosomes?

Answer 38

Large RNA subunit: catalyses the formation of the peptide bonds between amino acids - has 3 RNAs and lots of proteins
Small protein subunit: provides a scaffolding for the tRNA anticodons to be matched to mRNA codons - has one RNA and lots of proteins
The two subunits are separate until they associate with a mRNA for translation
The non-coding RNAs within the ribosome are what interact with tRNA and mRNAs.