Transcription & Translation Flashcards
Define cytosol
The aqueous component of the cytoplasm of the cell that glues the whole cell together
Define polycistronic and monocistronic DNA - are these in prokaryotes or eukaryotes
Polycistronic - prokaryotes - can code for multiple proteins
Monocistronic - eukaryotes - codes for a single protein (more specific)
Where is mitochondrial DNA inherited from?
Mother
How can mitochondria make their own proteins?
they have a small circular DNA and ribosomes
Are most mitochondrial DNA made in the mitochondria?
No, most are made in the nucleus and imported
Give 3 differences between RNA and DNA
RNA is single-stranded
RNA is less stable
RNA has a ribose sugar, fully hydroxylated
Uracil
What happens if a cell detects double stranded RNA and why?
An alarm is set off, because this indicates viral infection
What do the three types of RNA polymerase do?
RNA polymerase | = transcribes ribosomal RNA
RNA polymerase || = transcribes mRNA
RNA polymerase ||| = transcribes tRNA
What is a TATA box and what is its function?
A short run of A and T bases upstream of the start codon of a gene that act as a promoter sequence to signal the start of transcription. Transcription factors bind.
Why does a TATA box use A and T bases?
They have the lowest energy pairing so are easiest to unwind
What are transcription factors, what do they bind to and what binds to them?
Transcription factors are proteins that bind to the TATA sequence or other promoter to either promote or inhibit transcription, forming the transcription initiation complex. RNA polymerase || binds.
Do all mammalian genes have TATA boxes.
No, around 24% of genes.
What are CpG islands?
Stretches of DNA where at multiple points C is followed by G. They appear upstream of many genes and appear to have promoter activity.
How can the expression of a gene be switched off in terms of CpG islands?
CpG islands are unmethylated. To switch off the gene, cytosine can be methylated (CH3 added).
Give an example of when CpG methylation occurs
Cancer
X-inactivation by Xist
How is translation stopped?
A release factor binds to the stop codon. This is not yet understood. Transcription often carries on past the stop codon but is untranslated.
What is capping after transcription? Why?
The 5’ end of mRNA is capped using 7-methyl-guanosine via a chemical reaction with an unusual 5’ to 5’ linkage. This protects the mRNA from destruction by nuclease enzymes.
What is a poly-A tail and why is it added?
The 3’ end of the mRNA is cleaved after transcription at the poly-a-site sequence AAUAAA and up to 300 A nucleotides are added. This prevents degradation of DNA and may also have a role in transport to the cytoplasm
What is the usual splice site?
AG/G
What carries out splicing?
The spliceosome - a protein/RNA complex
What is a polypyrimidine tract?
A sequence of about 15 (mainly) C and T (U in RNA) bases upstream o the splice site, which gives context to the splice site
What is needed for splicing?
AG/G codons at the end/start of the two exons
Spliceosome
‘Context’
What are the regions before the first exon and after the last exon? Are these exonic or intronic?
The 5’ UTR and 3’ UTR - these are exonic because although non-coding, they are included in the mature mRNA.
Can exons change reading frames in the RNA?
yes
How do you make a gene construct in a mouse?
Add the desired DNA into some cultured cells. Some cells will express the DNA to make the protein.
Define transgene. What are the conditions for this to work?
A gene that has been transferred from one organism to another
Must be expressed in the same tissue, at an appropriate level and at the same developmental stage as the endogenous gene
Define Cis and Trans acting elements in DNA
Cis acting elements are DNA sequences in the structure of the gene e.g. a promoter region
Trans acting elements are proteins that bind to the DNA negatively or positively e.g. transcription factors
Which mammalian mRNAs are not spliced and polyadenylated?
Histones
What is the relevance of N-formylmethionine in bacterial proteins?
Bacteria add a modified version of N-formylmethionine into the first position in protein chains. This is not found in eukaryotic cells so is recognised as a foreign body and an immune response takes place.
How many amino acids are there?
20
What adds the amino acid to the tRNA molecule? What is different between different amino acids?
aminoacyl-tRNA synthetase using ATP and releasing AMP
Each amino acid has a different synthetase
What is charging?
An amino acid is added to a tRNA molecule using aminoacyl tRNA synthetase, energy from ATP is passed to the tRNA bonding is later used to make the peptide bond in the protein chain
What is wobble base pairing?
Each tRNA can recognise more than one codon because there are more codons than there are amino acids. The first base on the anticodon can recognise different bases in the THIRD base position in the codon. Also reduces the number of syntheses needed.
What are the E, P and A sites in ribosomes?
The A site receives tRNA carrying the next amino acid
The P site holds the tRNA attached to the growing polypeptide chain
The E site is the exit site
What is the start codon and amino acid?
AUG, methionine
What are the stop codons?
UAG, UUA, UGG
Why are AUG codons sometimes ignored by the ribosome?
They are in a ‘bad’ context
What gives energy to the movement of the ribosome along the RNA and the tRNA moving from the A site to the P site on the ribosome?
Hydrolysis of GTP to GDP
Define a polysome
An mRNA chain covered with many ribosomes, each transcripting a lengthening polypeptide chain
What do ribonucleases (RNAses) in the cytoplasm do?
Degrade mRNA - remove A residues from poly-A tail and remove the 5’ cap
How is mRNA degraded?
RNAses remove cap and poly-A tail and exonucleases remove nucleotides. Polysome structure offers protection against this.
Define nonsense mediated decay
A mutation causes a premature stop codon, so the mRNA downstream is not covered in ribosomes and is degraded quickly by exonucleases
Do eukaryotes or prokaryotes have 60:40S RNA:proteins? What is S?
Eukaryotes
S = Suedburg unit. Measures force required to pellet the molecule in a centrifugal field.
Do eukaryotes or prokaryotes have 50:30 RNA:proteins?
prokaryotes
What is the danger for mitochondrial ribosomes in terms of antibiotics?
Mitochondrial ribosomes resemble bacterial ribosomes so can be targeted by antibiotics.
What is dideoxy sequencing?
In a dideoxynucleotide, both OH on the sugar have lost the O. Dideoxyribose can’t make a phosphodiester bond because dehydration synthesis can’t occur, so the chain stops.
DNA sequencing is carried out using a dideoxynucleotide for each base at different times, and different length strands are made. Dyes are used to build up the DNA sequence.
What binds to and takes a protein to the ER?
A signal receptor particle (SRP)
How does a protein become incorporated into the ER?
ER receptors bind to the signal receptor protein that brings the protein to the ER and stimulate the formation of a transmembrane translocation channel through the ER. SRP dissociates and the peptide grows, with the N terminal being extruded into the lumen of the ER. The protein is modified as it grows. The signal sequence is cut off and the ribosome is released and dissociated. C terminus remains outside in the cytoplasm
What are chaperones?
Folding proteins
Why does an alpha helix usually form in transmembrane proteins?
It anchors the protein into the membrane (stop-transfer-membrane-anchor-sequence)
What are the three areas of the golgi?
Cis, Medial, Trans
What is glycosylation and where does it occur?
Sugars are added to proteins and some old sugars are trimmed back. In the Golgi
Why does the phosphorylation of proteins occur in the golgi? Where are phosphates added?
Phosphates are added to the manose group (a sugar added in the ER).
This signals to send proteins to the lysosomes, in vesicles.
How are proteins separated in the golgi?
Separated as to whether they are to be secreted into vesicles or not.
When are disulphide bridges formed?
RER
What is a multi-pass (polytopic) protein?
Can be threaded through the membrane multiple times due to variations in the translocation mechanism
What is prenylation? What signals it to occur?
Adding a lipid tail to the protein, so that it gets fixed in the membrane without being transmembrane.
Has a special sequence at the carboxyl terminal, which causes C terminal to be cleaved off and replaced with a fatty acid chain at the sulphur atom of a cysteine residue
What is interleukin 1B? Why is this different from most proteins after translation?
An inflammatory mediator secreted from cells after a heart attack, but has no signal sequence to direct it to the ER-Golgi route. Actual route is unknown but it is thought that its released through pores in the plasma membrane.
Why is blood clotting factor X111a different to most proteins in terms of targeting after translation?
It lacks an ER targeting signal sequence. It is unknown how it leaves the cell.
Why was it thought that insulin could be made in bacteria?
Insulin is not glycosylated
How is insulin made in yeast?
A fusion protein is made between yeast proteins and proinsulin, with a recognition site in the middle. The C peptide on yeast is shortened and mutated. The proteins are cleaved and the C peptide is removed from the chain
How is insulin made in humans?
It is made in the beta cells of the pancreas. It is not glycosylated (no signal sequence for sugars). It is made from 2 chains, A and B, connected by disulphide bridges formed in the ER. The C loop is cut off in the Golgi,
How are gene constructs & animal milk being used?
A transgene is incorporated into the mammary gland of lactating animals. Promoter sequence form the casein gene are used, including a few exons that don’t code for proteins, UTRs and poly-A tail. The DNA sequence for the desired gene is in the middle.
Define pharming
producing pharmaceutical products form farm animals
