Lecture 22 Flashcards
(22 cards)
Processing of rRNA
Most rRNA is transcribed by RNA polymerase I in the nucleolus. The 18S, 5.8S, and 28S rRNAs are initially transcribed as a single large precursor molecule called 45S pre-rRNA.
Nucleotide modifications
Happens at base or ribose group. Can involve adding methyl groups. Aims to increase fitness of ribosome.
Pre-rRNA is assembled with the ribosomal proteins
Forms one big ribonuclear protein
Cleavage of pre-rRNA into 18S, 28S and 5.8S rRNA
The 45S pre-rRNA is cleaved to produce the 18S, 5.8S, and 28S rRNAs. These are then assembled with ribosomal proteins into ribosomal subunits. The 5S rRNA is transcribed separately by RNA polymerase III in the nucleus and joins the large ribosomal subunit later.
Nucleotide cleavage at 5’ and 3’
Cleaved by RNA nucleases
Intron splicing on tRNA
Intron spliced and ends of remaining tRNA are ligated, forming anti-codon.
Processing of tRNA
Done by RNA Pol III. in nucleus
Addition of CCA nucleotides
Added to 3’ end. Constant site where a.a covalently binds.
Location of nucleotide modifications
Alterations at base/ribose group.
Processing of mRNA in pro vs euk
Most processed. In bacteria, transcription and translation occur in same region while in eukaryotes mRNA is transcribed in the nucleus, processed and sent to cytoplasm for translation
5’ capping
Occurs when transcript is ~25nt long. DURING transcription. 5’ triphosphate is hydrolysed by RNA triphosphate, leaving a diphosphate at 5’ end. Diphosphate ‘attacks’ alpha phosphorus of incoming GTP to form 5’ to 5’ linkage. 5’ to 5’ triphosphate linkage is only found at 5’ end. N7 nitrogen found at G base is methylated to form 5’ cap which attaches to nascent RNA via 5’-5’ linkage. Catalysed by 3 enzymatic reactions. Protects 5’ end degradation by phosphatases and nucleases and increases translation.
Polyadenylation of the 3’ end
Occurs AFTER transcription. As RNA is made, there is a specific sequence (AAUAAA) that acts as a cleavage signal. OH and poly(A) polymerase use ATP as substrate to add on adenine tail (about 250). Tail is made enzymatically, not encoded by DNA. Recruits proteins that can sit elsewhere on the mRNA which can stop cleavage enzymes from breaking mRNA. Increases translational efficiency. Occurs in most mRNA, except histone mRNA.
Histone processing
Have stem-loop structure followed by purine rich sequence (A/G) which recruits SiRNA and cleavage enzymes to cut mRNA forming mature histone mRNA.
Exploiting poly(A) tail
Used to isolate mRNA e.g production of oligo-dTs.
Splicing
> 90% human genes have exons (protein-coding) and introns (non-coding regions). Splicing removes introns and ligates exons together. Important as introns are non-coding and they’re long. After splicing, mature mRNA is formed.
Splice sites
Intron sequences have highly conserved sequences. At the 5’ end of the intron, there’s always ‘GU’, called the 5’ splice site. The 3’ splice site includes the polypyrimidine tract, which is a series of ~10 U/C bases, followed by some bases and ending in ‘AG’. Splicing occurs at the AG. The branch site is 20–50 nt upstream of the 3’ splice site and always has an ‘A’ in the middle.
Spliceosome
Large splicing complex composed of SnRNAs, proteins and pre-mRNA that is being spliced.
Incorrect splicing and disease
Mutations affecting splicing cause at least 15% of all genetic diseases e.g thalassemia arises from the defective synthesis of the B-globin gene resulting in bad haemoglobin. A mutation arises in DNA which is transcribed into mRNA. Truncated protein and mRNA causes degradation.
Alternative splicing
Multiple mRNAs from a single gene creates protein diversity from a limited no. of genes. Cell has a choice to include all exons or not. Just because its an exon, doesn’t mean that it’s always included in mature mRNA. If the exon may or may not be it is called alternatively spliced (>70% exons are). Introns are always excluded. If an mRNA has n exons, that can be alternatively spliced, there are 2^n different mature mRNA products.
Transcription and mRNA processing
These processes are coupled. RNA pol. II makes mRNA and is recruited by TFII which phosphorylates C terminal domain. This activates it, allowing RNA Pol II to recruit factors important for capping, adding poly(A) tail and splicing.
Exons in antibodies
If exon is included, an antibody that can bind to the membrane may be produced. If the exon is excluded, it may lose the capacity but still be functional.
mRNA transport
mature mRNA goes to cytoplasm from nucleus via nuclear pores. Number of accessory and transport proteins facilitate this. Through nuclear pore complexes.
