1 - Introduction to Transcription and Translation

Question 1

What are the features of a typical bacterium?

Answer 1

Lipid membrane - gram negative bacteria have a double layer of this.
Rigid cell wall.
DNA - circular chromosome as well as plasmids.

Question 2

What are plasmids?

Answer 2

Free circles of DNA inside the bacteria.
Can be transferred between bacteria.
Can encode antibiotic resistance genes.

Question 3

What is the circular chromosome attached to?

Answer 3

Attached to the membrane; it contains the most genes in the bacterium.

Question 4

How is eukaryotic DNA arranged?

Answer 4

Arranged in chromosomes which are wrapped around histone (protein) molecules.
It wraps around an octamer of core histones (H2A, H2B, H3, H4 - two copies of each) and is secured by a H1 protein molecule.

Question 5

Where in the nucleus are ribosomes assembled?

Answer 5

Nucleolus.

Question 6

Where does transcription occur?

Answer 6

Principally in the nucleoplasm, however it also occurs on ribosomes attached to the ER membranes and in the mitochondria.

Question 7

What are promoter elements?

Answer 7

Promoter elements are regions which encourage the transcription of genes.
Function of promoter elements is more readily understood in bacteria than in eukaryotes.

Question 8

What is a ‘TATA’ box?

Answer 8

Example of a promoter element.

Approximately 25bp long - exists prior to the start of transcription.

Question 9

How do promoter elements encourage transcription?

Answer 9

Transcription factors bind to the promoter element and form a protein complex - RNA polymerase binds to this complex.
RNA Pol II is involved in the transcription of mRNA molecules.

Question 10

What direction is RNA synthesised in?

Answer 10

5’ to 3’

Question 11

What is template strand also known as?

Answer 11

Antisense strand - it runs antiparallel to the transcribed RNA sequence. Hence, the ‘sense’ strand runs in the same direction as the RNA molecule.

Question 12

How does RNA differ to DNA? (4)

Answer 12

RNA is typically single stranded - double stranded RNA can be indicative of a viral infection.
RNA uses ribose sugars as opposed to deoxyribose.
RNA uses the base uracil instead of thymine (Uracil lacks the 5’ methyl group of thymine and can be formed from the deamination of cytosine).
RNA is less stable than DNA (due to the high activity of RNase enzymes).

Question 13

What are operons?

Answer 13

Operons are a mRNA sequences composed of linked genes - this means that multiple proteins can be coded from the same strand of mRNA.
The lecture slide states that this happens only in prokaryotes.
Note - Genes are arranged in operons in both prokaryotes and eukaryotes. In prokaryotes you would generally expect to see polycistronic mRNA, in eukaryotes you would expect monocistronic mRNA (which may be why the lecturer says ‘only in prokaryotes’).

Question 14

How is eukaryotic mRNA protected from degradation?

Answer 14

5’ cap - this is a methylated guanine molecule on the 5’ end of the mRNA sequence.
Poly-A tail - this is a stream of adenosine molecules occurring after the ‘cleavage site’. It is added through ‘polyadenylation’.

Question 15

How are introns removed from pre-mRNA?

Answer 15

Splicing of mRNA using ‘spliceosome’ enzymes.

Splicing enzymes are specific. Basic mechanism - they form a ‘lariat’ or a loop of intronic mRNA and remove it.

Question 16

What are ‘specific splice sites’?

Answer 16

Regions at the beginning and end of each intron that are recognised by spliceosome.
GU - 5’ end
AG - 3’ end

Question 17

Give an example of the use of alternative splicing of mRNA?

Answer 17

Synthesis of different types of myosin in different types of muscle.
Skeletal muscle and smooth muscle have characteristic differences which result from this alternative splicing.

Question 18

How many possible reading frames are there for mRNA, and why?

Answer 18

3 - codons consist of three letter units and can be read starting at different points.

Question 19

What is the basic structure of tRNA?

Answer 19

tRNA possesses a ‘clover leaf’ arrangement.
5’ end - D loop - anticodon loop - T loop - acceptor stem/3’ end
Amino acids attach to the 3’ end of tRNA.

Question 20

How are amino acids attached to tRNA?

Answer 20

Enzymes, called aminoacyl tRNA synthetases, charge amino acids to the tRNA molecule.
Each enzyme is specific for each tRNA.

Question 21

What is ‘wobble’ base pairing?

Answer 21

Non-Watson-Crick base pairing (involving the appropriate amino acid).
This reduces the total number of tRNA molecules/tRNA synthetases required.

Question 22

What codon initiates protein synthesis?

Answer 22

AUG codon and initiator tRNA carrying methionine (or, in bacteria, N-formyl methionine).
Note - initiator tRNA differs from a normal tRNA which carries methionine - it is recognised by initiation factors which bind it to the small sub-unit of the ribosome. This then travels along the mRNA until the AUG codon is encountered.

Question 23

Why is the coding region of mRNA covered with lots of ribosomes?

Answer 23

This enables multiple copies of the same protein to be formed at the same time and also prevents degradation of the mRNA by RNAses.
mRNA covered with multiple ribosomes is called a polysome.

Question 24

What are the different sites within the ribosome?

Answer 24

E - exit
P - peptidyl
A - aminoacyl

Question 25

What direction does the ribosome move in?

Answer 25

The ribosomes moves in a 5’-3’ direction along the mRNA.

Question 26

What are the ‘stop’ codons?

Answer 26

UAA, UAG and UGA

At these codons, release factors bind and the polypeptide chain is released from the ribosome.

Question 27

What is ‘nonsense mediated decay’?

Answer 27

A consequence of a premature stop codon in a mRNA sequence - mRNA becomes exposed to RNAses once the ribosome is released.

Question 28

What is the difference between prokaryotic and eukaryotic ribosomes?

Answer 28

Prokaryotic - 70S; 50S and 30S subunits.
Eukaryotic - 80S; 60S and 40S subunits.
(S units - Svedburg units)

Note - In the large subunit of eukaryotic ribosomes there is 28SrRNA present - this is measured to find out how much RNA is present in a sample of eukaryotic cells.

Question 29

What antibiotics bind to bacterial ribosomes?

Answer 29

Small ribosomal subunit - tetracycline, spectinomycin, hygromycin B and streptomycin.
Large ribosomal subunit - chloramphenicol, streptogramin B and erythromycin.

Note - whilst this seems vague, a question on this did come up on the final exam.