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