MCB 4: Transcription and Translation

Question 1

Define transcription

Answer 1

• the process of copying one strand of DNA into a complementary RNA sequence by the enzyme RNA polymerase

Question 2

What is always the initial product of gene expression?

Answer 2

• RNA

Question 3

What is a basal transcription complex?

Answer 3

• the bare minimum complex of proteins required for the initiation of transcription

Question 4

What do you need for basal transcription complex to form?

Answer 4

• particular sequences are required in the promoter region:
• TSS (at +1)
• TATA
• TFBS

Question 5

Describe the first base of a gene promoter, TSS

Answer 5

• first base, written as +1

positions with a negative value (anything before TSS) is upstream and positions with a positive value are downstream of TSS

Question 6

Describe TATA in the promoter region

Answer 6

• resides around -10 upstream
• DNA sequence in this region usually resembles TATA, not completely, but dominated by T and A

Question 7

Describe TFBS as a gene promoter

Answer 7

• transcription factor binding sites (TFBS) are specific sequences that transcription factors bind to and module gene expression
• they are dispersed throughout the promoter and control the rate of transcription
• can also be found at enhancers, regions further away from the transcription start site where transcription factors and other proteins bind, where they can also influence gene expression

Question 8

Describe how the basal transcription complex is formed

Answer 8

1. TFIID, a complex of proteins that contain TATA Binding Protein (TBP) and TBP Accessory Factors (TAFs), binds to TATA, causing the DNA to partially unwind
   - this allows more contact with bases
   - unwinding is asymmetric with respect to the TBP-TATA complex is unidirectional
2. Next, both TFIIA and TFIIB bind
   - TFIIB is able to bind to both TFIID and RNA polymerase, so it is very important to complex formation
3. TFIIB allows RNA polymerase to bind, with TFIIF attached
4. Finally, TFIIE, TFIIH and TFIIJ bind
   - TFIIH specifically promotes further unwinding of the DNA helix to facilitate RNA synthesis
   - Now RNA polymerase II can be phosphorylated, which activates it to begin transcription at a basal (low) level
5. The addition of transcription factors modulates this transcription level
   - they interact with each other, RNA polymerase and also other proteins which might modify the chromatin state

Question 9

Describe RNA capping

Answer 9

• a 5’-cap, 7-methylguanosine (m⁷-G) is added at the 5’ end of the pre-mRNA
• note the unusual 5’-to-5’ linkage
• it is performed by three enzymes associated with RNA polymerase II C-terminal domain (CTO):
• Phosphatase: removes a phosphate from the 5’ end of the nascent RNA transcript
• Guanyl transferase: adds a GMP with a 5’-to-5’ linkage
• Methyl transferase: adds a methyl group to guanosine

Question 10

What is the function of RNA capping?

Answer 10

• protects the mRNA from degradation
• helps the mRNA to be effectively spliced and exported out of the nucleus
• helps during mRNA translation

Question 11

Describe the process of RNA splicing

Answer 11

See both images

The molecular machinery involved is known as the spliceosome and is a protein complex so large it is considered an organelle that forms from protein and RNA/protein complexes (snRNPs) as follows:

1. BBP (Branch-point Binding Protein) and U2AF recognise and bind to the branch point in the intron and U1snRNP recognises and binds to the 5’ splice site by forming base-pairs.
2. U2snRNP replaces BBP and U2AF by binding to the branch point.
3. U4/U6snRNP and U5snRNP bind to the 5’ splice site as well leading to several RNA rearrangements that break up the U4/U6snRNP and form a loop in the intron.
4. U6snRNPis now the only protein at the 5’ splice site, having replaced U1snRNP and U4snRNP.
5. A conserved Adenine nucleotide at the branch point attacks the 5’ splice site and cuts the sugar-phosphate backbone of the RNA.
   - The G residue from the start of the intron forms a phosphodiester bond with the A residue to form the lariat shape.
6. The spliceosome brings the two exons together, allowing the 3’ -OH group of one exon to react with the 5’ end of the other, and are ligated.
7. The 3’ splice site is then cleaved, allowing the lariat and associated snRNPs to be released and degraded.

Question 12

What sequences signals the 5’ splice site in RNA splicing?

Answer 12

• AGGU

Question 13

What sequence signals the 3’ splice site in RNA splicing?

Answer 13

• AGG

Question 14

How is the 3’ end of the pre-mRNA processed?

Answer 14

• a series of proteins and enzymes cleave the pre-mRNA and add a poly-A tail (called cleavage and polyadenylation)

Question 15

Describe the major steps of the 3’ end processing of pre-mRNAs

Answer 15

Question 16

Define translation

Answer 16

• the process by which the sequence of nucleotides in an mRNA molecule directs the incorporation of amino acids into a protein on a ribosome

Question 17

Define 5’ UTR, 3’ UTR, CDS, stop codons, start codons

Answer 17

Question 18

What is a codon?

Answer 18

• a sequences of three nucleotides that corresponds with a specific amino acid or stop signal during protein synthesis

Question 19

Describe the structure of tRNA

Answer 19

• a single-stranded RNA molecule that folds into a specific 3D structure

Question 20

How are amino acids loaded onto tRNA?

Answer 20

• at the 3’ end, amino acids are loaded onto tRNA by specific enzymes called: aminoacyl-tRNA synthases
• the anticodon matches the codons in mRNA

Question 21

What are the three stages of translation in eukaryotes? Briefly describe them

Answer 21

1. Initiation:
   - the assembly of the ribosome with the first tRNA at the start codon
   - assisted by eukaryotic initiation factors (eIFs)
2. Elongation:
   - the transfer of amino acids to the growing polypeptide and the movement (translocation) of the ribosome to the next mRNA codon
   - assisted by eukaryotic elongation factors (eEFs)
3. Termination:
   - the release of the polypeptide upon reaching a stop codon
   - assisted by eukaryotic release factors (eRFs)

Question 22

Describe the initiation stage of translation

Answer 22

Question 23

Describe the elongation stage of translation

Answer 23

Question 24

Describe the termination stage of translation

Answer 24

Question 25

What are the three types of gene mutation?

Describe what they are

Answer 25

• deletion:
• a nucleotide base is deleted
• ACTG becomes ACG
• substitution:
• a nucleotide base is replaced by another
• ACTG becomes ACGG
• insertion:
• a nucleotide base is added as extra
• ACTG becomes ACATG

Question 26

What is a silent mutation?

Answer 26

• if two different triplet codes translate into the same amino acid
• the polypeptide chain remains unchanged

Question 27

What is a scenario where the mutation in the DNA sequence might not affect the protein?

That is not a silent mutation?

Are there any limitations of this?

Answer 27

• if a mutation occurs within an intronic region, it is less likely to affect the protein
• but if thre mutation affected the 5’ or 3’ splice site or the conserved sequence within the intron, then the splicing process could be affected
• this could affect which exons are included in the cDNA
• therefore, what amino acids will make up the protein

Question 28

What is a frameshift within the coding sequence?

What type of mutation causes this?

Answer 28

• insertion or deletion of a nucleotide within the coding sequence will cause a frameshift
• this affects the codon sequence
• therefore the amino acids
• may also generate a premature stop codon

Question 29

What is a missense mutation?

What are the potential consequences of it?

Answer 29

• If the mutation changes the amino acid sequence it is known as a missense mutation
• This could affect protein structure
• for example, changing the charge on an amino acid side group, affecting the way that the protein folds
• It could affect whether a substrate or ligand still binds to the protein, or even create a new binding site.
• It could change an enzymatic function that protein carries it out, making it worse or better, depending on the change.

Question 30

What is a nonsense mutation?

What are the consequences of them?

Answer 30

• If a premature stop codon is created, this is known as a nonsense mutation
• This could result in a truncated protein, which may partially function, or function differently, or could result in degradation of the mRNA, preventing this protein from being expressed.

Question 31

What are chromosome mutations?

Answer 31

• Mutations can also be larger chromosomal changes, affecting a section of the chromosome that could contain one or more genes within it.
• There are four types of chromosome mutation

Question 32

What are the four types of chromosome mutation?

Answer 32

• duplication:
• a region is duplicated: resulting in extra copies of a gene
• deletion:
• a region is deleted: resulting in less copies of a gene and a lost of heterozygosity
• inversion:
• a region is inverted or flipped: this could result in a change in the regulation of gene expression
• translocation:
• a section of chromosome is switched with another
• this can also lead to a change in the regulation of gene expression

Question 33

Explain colloquial variant nomenclature

Answer 33

This involves single letter amino acid codes and numbers to denote substitutions at certain positions. For example, R117Hdenotes Arginine at position 117 on the polypeptide chain being replaced by a Histidine. It can also represent creations of premature stop codons with the letter ‘X,’ e.g.: W1282X denotes a Tryptophan at position 1282 being replaced by a stop codon.

Deletions can be represented in a number of ways such as Delta F508, which denotes the deletion of Phenylalanine at position 508, or by 2148delA which denotes the deletion of an adenine base at position 2148 of the DNA sequence.

Problems with this nomenclature arise due to the lack of clarity regarding the change in DNA which results in the particular change in amino acid. It is also impossible to represent intronic mutations using this method and the letters representing the bases (A, C, G and T) also represent amino acids, which could cause confusion.

Question 34

Explain standard mutation nomenclature

Answer 34