MGD S5 - Transcription and Translation

Question 1

Give a very basic description of the function of transcription and translation

Answer 1

• Transcription: DNA converted to mRNA, nucleotide to nucleotide, “copying the code”, simple, in nucleus
• Translation: mRNA converted to protein, nucleotide to amino acid, “changing the language” - code translated, more complex, 4 letter to 20 letter code, adaptor molecule needed, in cytoplasm

Question 2

What is a gene?

Answer 2

• A stretch of DNA at a chromosomal locus (specific location) - “Recipe” for a protein code and regulation - There are ~25,000 genes, 2x25,000 in every cell (not expressed all the time)

Question 3

What is the difference between heterochromatin and euchromatin?

Answer 3

• Heterochromatin: dark, solenoid 30nm fibre, genes not expressed - Euchromatin: light, beads on a string, genes expressed

Question 4

What are the different types of RNA?

Answer 4

• mRNA (messenger RNA) - rRNA (ribosomal RNA) - tRNA (transfer RNA) - miRNA (micro RNA) - noncoding RNA: regulate their counterpart

Question 5

What is involved in making DNA?

Answer 5

• Needs an enzyme: DNA polymerase - Needs activated substrates: dNTPs - Needs a template: DNA - 3 stages process: initiation, elongation, termination

Question 6

What is involved in making mRNA?

Answer 6

• Needs an enzyme: RNA polymerase - Needs activated substrates: NTPs - Needs a template: DNA - 3 stages process: initiation, elongation, termination

Question 7

What is involved in making a polypeptide / translation?

Answer 7

• Needs an enzyme: ribosome - Needs activated substrates: amino acids - Needs a template: mRNA - 3 stages process: initiation, elongation, termination

Question 8

Give an overview of DNA replication

Answer 8

• Initiation: recognition of origin of replication, initiation proteins, DNA polymerase - Elongation: 5’ to 3’ chain growth - Termination: when replication forks meet

Question 9

Give an overview of transcription

Answer 9

• Initiation: promoter recognition, transcription initiation factors, RNA polymerase - Elongation: 5’ to 3’ chain growth - Termination: sequence dependent - Making a polynucleotide (mRNA) - (rNMP)n + rNTP ➡️ (rNMP)n+1 + PPi

Question 10

What is a transcription unit?

Answer 10

• A gene - Genes go in different directions

Question 11

What is the significance of the promoter in initiation?

Answer 11

RNA polymerase must recognise the appropriate point on which to start transcription and the strand of DNA to transcribe. The signals in DNA that RNA polymerase recognises are called promoters. Promoters are sequences in DNA that determine the start point and the frequency of transcription

Question 12

What is the importance of how the transcription factor binds to the DNA in initiation?

Answer 12

Determines the direction of translation, and therefore the direction of the gene

Question 13

Describe initiation in transcription

Answer 13

Promoters recruit the RNA polymerase to the transcription start site on DNA. Promoters are usually located in front (upstream) of the gene that is to be transcribed - where promote sits on sequence is where transcription starts. Must be 5’ to 3’ for transcription factor to bind

Question 14

Describe the difference between promoter sequences and regulation in eukaryotes and prokaryotes

Answer 14

• Eukaryotes: TATA box at -30, variety of upstream sequences, complex regulation - Prokaryotes: Pribnow box at -10, upstream sequences at -35, “simple” regulation

Question 15

Describe the process of elongation in transcription

Answer 15

• The process by which single nucleotides are added to the growing RNA chain - Ribonucleotides bind to an entry site on the RNA polymerase. If the incoming ribonucleotide matches the next base on the DNA a template, the incoming ribonucleotide is transferred into the polymerase active site and a new phosphodiesrer bond is formed. If not: process repeated until correct ribonucleotide is found - mRNA 5’ to 3’, template 3’ to 5’

Question 16

What is significant about the DNA coding strand in transcription?

Answer 16

Sequence identical to mRNA sequence. Both complementary to template/non-coding strand

Question 17

Describe termination in transcription

Answer 17

• Catalysed by multiple mechanisms - At the end of a transcriptional unit, the RNA polymerases terminate RNA synthesis at defined sites

Question 18

Summarise transcription

Answer 18

RNA polymerase binds to the transcription factor complex in the promoter region and to the DNA. The helix unwinds within a region near the start point of transcription. DNA strand separation occurs. Synthesis of RNA transcript is initiated, and the RNA transcript is elongated, copying the DNA template. The DNA strands separate as the polymerase approaches and rejoin as the polymerase passes

Question 19

What is an open reading frame (ORF)?

Answer 19

A portion of a DNA molecule that, when translated into amino acids, contains no stop codons

Question 20

Describe in RNA processing how pre-mRNA is transformed to mature mRNA

Answer 20

• Capping: at 5’ end, a 5’ cap. Protection against degradation - Tailing or Polyadenylation: at 3’ end a PolyA tail is added. Protection against degradation - Splicing: in the middle. Removes introns. Sequence dependent

Question 21

Describe capping

Answer 21

• A 5’-5’ linkage is created (extra nucleotide being added on) - Provides protection against degradation - Plays role in translation

Question 22

Describe nucleases

Answer 22

Degradation of nucleic acids - Endonuclease: breaks within polynucleotide. Non-specific (random cutting) or specific (recognises specific sequence and makes cut) - Exonuclease: degrades polynucleotide from end. 5’ specific or 3’ specific

Question 23

Describe polyadenylation/tailing

Answer 23

See image

Question 24

Describe splicing

Answer 24

Introns cut out - highly sequence-dependent. Leaves mature mRNA. Happens in all cells. Can go wrong

Question 25

Describe mature mRNA

Answer 25

See image

Question 26

Where does translation occur?

Answer 26

In the cytoplasm

Question 27

Describe the main different types of RNA and what abundance they appear in

Answer 27

• rRNA (ribosomal). RNA polymerase I. Few kinds. Many copies of each. Over 80% - mRNA (messenger). RNA polymerase II. 100,000s kinds. Few copies of each. ~2% - tRNA (transfer). RNA polymerase III. ~100 kinds. Very many copies of each. ~15%

Question 28

Describe rRNA/ribosomes in eukaryotic and prokaryotic cells

Answer 28

• Prokaryotes: 3 rRNAs and 56 proteins. 30S + 50S subunits. 70S ribosome - Eukaryotes: 4 rRNAs and 82 proteins. 40S + 60S subunits. 80S ribosome - Ribosomes are not specific - can translate any kind of mRNA

Question 29

Describe the genetic code

Answer 29

• From 4 letter “DNA language” to 20 letter “protein language” - Triplet - 64 possibilities, we need 20 - Non-overlapping and “comma-less” (no gaps) - “Changing the language” results in 5’ to 3’ template read through producing N (amino) and C (carboxy) polypeptide chain extension - Adaptor molecule required is tRNA - Initiation: AUG (methionine) - Termination: UAA, UAG, UGA. Not amino acid: tells translation machinery to stop

Question 30

Describe tRNA

Answer 30

• Clover structure: complementary sequences of antiparallel RNA strands form the loop - LH image is of uncharged tRNA. RH: amino acyl tRNA, which is charged with amino acids connected. Different names - Anticodon complementary to codon - Methionyl tRNA anticodon 5’CAU recognises mRNA codon 5’AUG. Alanyl tRNA anticodon 5’IGC, the codons 5’GCU, 5’GCC, 5’GCA. Base I is inosine: aspecific, can recognise all bases apart from G - 5’ base of anticodon and hence 3’ base of codon is the “wobble” position, allowing a single tRNA species to recognise more than one codon

Question 31

Give an overview of translation

Answer 31

• Initiation: AUG codon recognition, “special” methionyl tRNA ribosome - Elongation: N to C chain growth, amino acyl tRNAs - Termination: stop codons

Question 32

What is the function of amino acyl tRNA synthetase?

Answer 32

Adds amino acids to tRNA to make amino acyl tRNA. Energy dependent

Question 33

Describe initiation in translation

Answer 33

• Synthesis of a protein is initiated at the first AUG (methionine) codon in the mRNA - 40S subunit of the ribosome forms a complex with initiation factors, which binds to a Met-tRNA complex. Directed to 5’ end of mRNA, to 5’ cap. Complex scans mRNA until it locates the first AUG codon, using ATP to power this process. - 60S subunit then binds to the small subunit/Met-tRNA/mRNA complex, hydrolysing GTP and releasing initiation factors in the process

Question 34

Describe elongation in translation

Answer 34

• mRNA read codon by codon from 5’ to 3’, while polypeptide chain growth is from amino to carboxy terminus - Begins with binding of charged tRNA to A site of ribosome. Met-tRNA recognises AUG on P site. Binding of Met-tRNA, hydrolysing GTP. -Once correct charged tRNA molecule has been delivered to the A side of the ribosome, peptidyl transferase activity of the ribosome catalyses the formation of a peptide bond between the amino acid in the A site and the amino acid in the P site - The ribosome is then moved one codon down to the mRNA (towards 3’ end) and tRNA in A site moved to P site. Whole process recycles for addition of next amino acid

Question 35

Describe termination in translation

Answer 35

When A site if ribosome reaches on of stop codons of mRNA. Protein that is attached to last tRNA molecule in the P site is released. Energy-dependent reaction catalysed by hydrolysis of GTP, which transfers a water molecule to end of protein, thus releasing it from tRNA. After release of newly synthesised protein, ribosomal subunits, tRNA and mRNA dissociate from each other, setting stage for translation of another mRNA

Question 36

What is polyribosome/polysome?

Answer 36

mRNA template with lots of actively translating ribosomes moving along

Question 37

Describe features of transcription and translation in bacteria

Answer 37

Bacteria have: - Different transcription factors - Single RNA polymerase: we have 3 types because no nucleus - Coupled transcription-translation - No post-transcriptional processing: no introns - Short lived mRNAs - Simpler ribosomes - Distinctive translation mechanism - Different translation factors

Question 38

Describe some antibiotics

Answer 38

• Tetracycline: binds 30S ribosomal subunit, blocks aminoacyl tRNA binding - Choloramphenicol: binds 50S ribosomal subunit, affects peptidyl transferase action - Erthyromycin: binds 50S ribosomal subunit, prevents translocation (A site remains unoccupied)