Nucleic Acids - Transcription, Translation and Targeting Flashcards
What is the template (antisense) strand?
The strand which is transcribed
What are the 3 mammalian RNA polymerases?
- Polymerase I transcribes ribosomal RNA
- Polymerase 2 transcribes mRNA, microRNAs and a variety of non-coding RNAs
- Polymerase 3 transcribes tRNA (transfer RNA)
What is not needed in transcription compared to DNA replication and why?
An RNA primer is not needed as it is single stranded
What is a promotor element?
region of DNA upstream of a gene where proteins e.g. RNA polymerase, transcription factors bind to initiate gene transcription
What is a TATA box?
A promotor element - a short run of T and A bases that can vary slightly from gene to gene .
It may be that Ts and As are used because they form the lowest energy base-pairs and so are the easiest to unwind.
How does the Tata box act as a promotor?
- The gene has a TATA box upstream and then 25 base pairs until transcription start site (very close)
- Transcription factors bind (several proteins form a complex)
- RNA polymerase bind II binds to the transcription factors
- Some transcription factors disassociate and transcription factors
What are characteristics of promotor elements situated further away?
- several kilobases away from transcription start site
- 1 to 3 kb stretch of proximal enhancers/silencers (some may be involved in tissue-specific and regulated transcription)
- Followed by minimal core promotor sometimes including a TATA box
- 25 base pairs
- transcription start site
Which group does methylation of cytosine add? What is produced?
CH3 is added to form 5-methyl cytosine in DNA
What does 5-methyl cytosine pair with in DNA and when does it most commonly occur?
Still pairs with G and most commonly occurs to the C when it is followed by a G in the DNA sequence = CpG (C phosphodiester link G)
What are CpG islands?
- sequences of a few hundred bp of DNA which have a particularly high density of CpG dinucleotides
- CpG islands are present about 1 kilobase upstream of the transcription site of mammalian genes
Methylation of C in active genes?
Active genes (genes that are being transcribed) = CpG island dinucleotides unmethylated whereas Cpgs elsewhere in the gene are methylated
Methylation of C in inactive genes?
CpG island dinucleotides are methylated and CpGs present elsewhere in the gene are methylated
What are the two steps needed to generate mature mRNA from pre-mRNA?
1) Cleavage and polyadenylation
2) Splicing out of introns
How does cleavage and addition of a polyA tail occur?
1) Pol II generates a pre-mRNA. In the region of DNA encoding the 3’ UTR there will be one or more polyadenylation sequences. One of these likely to be used more.
2) In eukaryotes, transcription continues past the point where polyadenylation sequences are present. Transcription termination in eukaryotes is poorly understood but many kilobases of extra sequence may be copied from DNA in to RNA
3) The mRNA is cut a short way downstream (-30 nucleotides) of the polyadenylation sequence (AAUAAA in RNA)
4) A polyA tail is added
Describe the 5’ cap and poyA tail on eukaryotic mRNA
Capping nucleotide found on the 5’ end (stabilises mRNA) connected to the 5’ UTR (untranslated region) which is connected to the coding sequence followed by the 3’ UTR and a polyA tail added at the end (except histone mRNAs).
What type of RNAs can bacteria produce that eukaryotes cannot?
Polycistronic mRNAs (encodes two or more proteins)
What does the lac operon produced by E.coli do?
Three coding sequences within the mRNA
- lacZ sequence produces an enzyme which breaks down lactose to monosaccharides
- lacY seq produces an transporter necessary for the uptake of lactose
- lacA seq produces an acetylation enzyme whose biological function is not completely understood
What is splicing?
The removal of intronic sequences from the pre-mRNA
How many nucleotide pairs and exons are there in the human B-globin gene?
2000 nucleotide pairs, 3 exons
How many nucleotide pairs and exons are there in the human Factor VIII gene?
200,000 nucleotide pairs, 26 exons
How does splicing occur?
- Introns are removed by the spliceosome
- A lariat is formed from the intron and cut out
- Alternative splicing (including different exons in different mRNAs) can give subtly different mRNAs in related tissues
What is the difference between prokaryotic mRNA and eukaryotic mRNA?
Prokaryotic mRNA does not have a polyA tail or a 5’cap, it can also be polycistronic.
How can you test whether all of the control elements in DNA necessary for the proper regulation of transcription are present? (e.g. B-globin gene)
- make DNA constructs covering globin gene and flanking sequences
- inject into mouse oocytes, some DNA will integrate at random sites into the mouse genome within the oocytes.
- Allow the recombinant oocytes to develop into transgenic mice
- If all of the regulatory info is correct, the construct will always work where it is integrated (expresses at the right level, right stage of development and in the right tissues)
How many natural amino acids are there in the body?
20
What is a codon?
3 bases that code for an amino acid
*more than one codon can code for an amino acid
What are the three stop codons?
- UAA
- UAG
- UGA
What is the universal go codon (Methionine)?
AUG
How is protein synthesis initiated (translation)?
- Initiator tRNA carries the amino acid methionine (or in bacteria N-formyl methionine) to AUG codon
- Initiator Met-tRNA differs from the normal tRNA that carries methionine
- initiator Met-tRNA is recognised by eukaryotic initiation factors that bind to the small subunit of the ribosome.
How does the small subunit of the ribosome initiate translation?
- Small subunit binds to the mRNA and moves along the 5’ UTR (scanning) until it reaches the initiating AUG
- The large subunit binds and translation starts
*often the initiating AUG is the first AUG in the mRNA reading from the 5’ end
*In some mRNAs, one or more of AUG codons in the 5’ UTR are overlooked before initiating AUG is reached. The initiating AUG will be in good context to initiate translation
What happens once the large ribosome subunit binds and translation is initiated?
- The ribosome moves along the coding sequence of the mRNA and the protein is synthesised.
- Additional ribosomes can engage with the mRNA before the first ribosome has completed translation
What is a polysome?
An mRNA with multiple ribosomes attached
What happens once the ribosome reaches the stop codon?
- Release factor binds to the A site, polypeptide chain is released from the ribosome.
- The ribosome dissociates and the protein is released
- If a premature stop codon is generated by mutation, the coding mRNA downstream of the premature stop codon will no longer be covered with ribosomes.
- The exposed region of the mRNA that isn’t covered with ribosomes may be chopped up by RNAses
- The mRNA degradation that occurs is called ‘nonsense mediated decay’
How are amino acids attached to tRNA before they can be incorporated into proteins?
They are esterified to tRNA
How are amino acids attached to tRNA before they can be incorporated into proteins?
They are esterified to tRNA
What is the structure of tRNA?
- Clover leaf structure
- The anticodon which hydrogen bonds with the codon in the mRNA is at the end of one loop
- At the 3’ end of the molecule the amino acid is attached
If anticodon 5’ GAA 3’ is present on tRNA what is the codon of RNA that will bind?
3’ UUC 5’ as it binds in an antiparallel fashion
If anticodon 5’ GAA 3’ is present on tRNA what is the codon of RNA that will bind? (Phenylalanine)
3’ UUC 5’ as it binds in an antiparallel fashion
What do aminoacyl tRNA synthetases do?
Esterify the correct amino acid onto each tRNA. There is a specific enzyme for each tRNA so the correct amino acid is added to the correct tRNA.
What does the yeast tRNA anticodon for phenylalanine 5’-GAA-3’ recognise as a codon which is unexpected? How is this beneficial?
- 3’-UUU-5’ (a non-Watson-Crick base pairing)
- Reduces the number of tRNA molecules and synthetases that the cell needs to produce
What are the three sites on an assembled ribosome?
- E (exit) site
- P (peptidyl) site
- A (aminoacyl tRNA) site
What is the difference in structure between prokaryotes and eukaryotes?
- Prokaryotic ribosomes are 70s which have a 50s large subunit and a 30s small subunit
- Eukaryotic ribosomes are 80s which have a 60s large subunit and a 40s small subunit
Why is having a different ribosomal structure in prokaryotes and eukaryotes important clinically?
Different antibiotics bind to bacterial ribosomes and inhibit protein translation
What are the S units in ribosomal structure?
Svedberg units - measure of the rate of sedimentation under centrifugal force
Apart from the cytosol and the ER where else are ribosomes found in the cell?
In the mitochondrial matrix on the cristae
What are certain antibiotics that target the small ribosomal unit in prokaryotes?
- tetracycline
- spectinomycin
- hygromycin B
- streptomycin
What are certain antibiotics that target the large ribosomal unit in prokaryotes?
- chloramphenicol
- erythromycin
- streptogramin B
Where is pre-mRNA processed into mature mRNA?
In the nucleoplasm and then it travels through nuclear pores which span the two nuclear membranes and is translated in the cytosol
what is the cytosol?
The thick liquid or gel surrounding the organelles
What is the cytoplasm?
the cytosol and the organelles but not the nucleus
What is the function of the ER and Golgi apparatus?
packaging and secretion of proteins that are due to be released from cells and the assembly of proteins embedded in membranes such as the plasma membrane.
what is the role of lysosomes?
They are full of degradative enzymes, they degrade certain molecules imported into the cell and damaged cell components.
- The degradative enzymes are delivered to the site via the ER and golgi apparatus
How are most mitochondrial proteins produced and transported ?
Encoded by nuclear genes then imported into the mitochondria afterwards.
*There are ribosomes present in the mitochondria for translation
How do cytosolic ribosomes become membrane bound ribosomes?
- Attach to an mRNA that happens to encode a secretory protein
- translate mRNA and the first bit of protein produced is a stretch of hydrophobic amino acids called the signal sequence
- Signal recognition particle (SRP) binds to the signal sequence causing the ribosome to dock to the SRP receptor on ER
- protein fed into a translocation pore on the ER
- From now the newly synthesised protein is fed through the pore into the ER as it is translated
- Usually the amino acid terminal signal sequence is cut off as a translocation of the bulk of the protein occurs
How is a secretory protein synthesised in the ER?
Translation through translocation channel and signal sequence is cut off. Once translocation is complete, the secretory protein is inside the ER and the translocation pore closes.
How are transmembrane proteins produced by the ER?
- Translation/ translocation begin as a secretory protein until a stretch of approximately 20 consecutive hydrophobic amino acids (a transmembrane domain) is made.
- The reminder of the protein is synthesised by the ribosome but is not fed into the ER
- The hydrophobic stretch of amino acids leaves the translocation pore and embeds itself into the membrane.
- The ribosome finishes translation of the protein, releasing the C terminus into the cytosol.
What are modifications made to the secretory pathway?
- ER proteins are folded, disulphide bridges are formed if present
- sugar side chains added if appropriate signals are present on proteins
- Some ER sugars are trimmed back from the trees and replaced with sugars added in the golgi apparatus
How are disulphide linkages made between cysteine residues?
Cysteine residues contain a HS group, these lose their H+ group to form a link between the sulphides.
What are examples of modifications that can happen when a transmembrane protein (e.g. insulin/LDL receptor) reaches the plasma membrane?
- Protein is anchored in the plasma membrane by the transmembrane domain
- Outside of the cell, trees of sugar units may have been added (‘glycosylation’)
- Disulphide bridges may have been formed
What is prenylation?
Addition of hydrophobic compounds to proteins or chemical compounds
What is the process of prenylation?
- The protein is made in cytosol with a particular amino acid sequence at the carboxyl terminus
- The presence of this sequence results in part of the carboxyl terminus being cleaved off and a fatty acid chain being transferred to the sulphur atom of a cysteine residue
- The fatty acid tail is believed to bury into the lipid bilayer
What is an example of the sequence construct made to express human proteins in the mammary gland?
1) locus control of the B globin gene to insulate the transgene from the influence of other DNA sequences
2) 3kb of promoter sequences from the goat casein gene to ensure expression in the mammary gland
3) exons from the goat casein gene that contain untranslated regions
4) cDNA coding the protein to be expressed in and harvested from the milk
5) exons from the goat casein gene that contain untranslated regions
6) polyadenylation sequences from goat casein gene
What is an example of a product producing human therapeutic proteins in cows and goats?
ATryn (Antithrombin Recombinant)
What is a popular way of generating insulin for patients with diabetes?
Yeast can make human insulin
