Module 6/7 - From RNA to protein ~ further reading Flashcards
Drosophila: how is RNA splicing used and how many proteins can a Drosophila gene form?
Used to determine the sex of the fly
One gene has been found to form 38,000 different proteins
What are the ways that RNA splicing can be used?
Used to make a protein either functional or nonfunctional:
* Transposase for the P element in Drosophila is turned off in somatic cells and on germ cells so transposition can occur in the genome without harming somatic cells)
Producing specialised forms of a protein:
* Tropomyosin, the protein that covers myosin binding sites to prevent contraction and allow relaxation, has several specialised forms throughout the body
How is RNA splicing regulated?
Positively:
Regulatory molecule instructing splicing machinery where splicing needs to occur
Negatively:
Regulatory molecule that prevents splicing machinery from gaining access to a splice site. This, however, may cause further splicing as strongly blocking a splice site will expose a splice site that is bonded to weakly
The definition of a gene
Has had to be modified to suit the fact that one gene may produce multiple different proteins.
The definition now states that a gene is any DNA sequence that is transcribed as a single unit and encodes one set of closely related polypeptide chains (protein isoforms)
A section of DNA that produces vastly different proteins is said to have genes overlapping: one for each vastly different protein produced
Sex determination in Drosophila: how does determination occur, what are the processes that produce males, what are the processes that produce females, and what are the flaws in this process?
Occurs due to the ratio of X chromosomes and autosomes: 2:2 produces females, 1:2 produces males
In a 1:2 ratio, no splice sites are blocked and nonfunctional proteins are produced from the sex-lethal (Sxl) and transformer (Tra) genes which result in the doublesex (Dsx) genes producing a protein with 550 amino acids (400 general and 150 male-specific) that repress female genes, resulting in male development
In a 2:2 ratio, an Sxl splice site is blocked, forming a functional Sxl protein that binds to a splice site in the Tra gene, which produces a Tra protein that, in conjunction with a Tra2 protein, activates a Dsx splice site, producing a 430 amino acid protein (400 general and 30 female-specific) repressing male genes, resulting in female development
The continual production of nonfunctional proteins in the male development of Drosophila is extremely wasteful, scientist theorise that this is caused by an ancient method of control that was used when RNA was the predominant biological molecule and so this method had to use lots of RNA-RNA interactions to work
How do B lymphocytes go from producing proteins acting as antigens to producing proteins acting as antibodies?
The B lymphocytes produce antibodies that anchor into the membrane and act as antigens, waiting to be activated
When the antigen is activated, the protein production is changed at the C-terminus end of the protein: in the antigen-antibody, there is a long string of hydrophobic amino acids on the terminus, resulting in a membrane-bound form; however, in the secreted antibody, the C-terminus is changed to a shorter sequence of hydrophilic amino acids which result in a secreted form.
How the difference in splicing the antibody gene in B lymphocytes changes whether it is membrane-bound or secreted
There are two Poly-A sequences on the RNA transcript. These sequences are where splicing may occur and this is regulated by a subunit of a protein, CstF, which binds to G/U-rich sequences and promotes splicing
When the antigen is not activated, the first Poly-A sequence is ignored and the RNA is spliced at the second Poly-A sequence, producing the longer, membrane-bound form
When the antigen is activated, CstF concentration is increased and so the first Poly-A sequence is recognised and splicing occurs there, forming the shorter, secreted form of the protein.
ADARs: what are they and what do they do?
Adenosine deaminases acting on RNAs
Convert adenosine into inosine
Why does RNA editing exist?
No conclusive answer but a few theories:
- Fixing “mistakes” in the genome
- Slapdash way of having one gene make slightly different proteins
- Evolved as a defence from retroviruses, extensively editing the viral genome makes mutates the genome and keeps the mRNA in the nucleus for degradation
How does HIV get its viral RNA that needs introns out of the nucleus?
RNA is fully spliced, producing Rev, a protein that binds to a Rev response element (RRE) in the RNA sequence. This, along with other important viral proteins, is used to interact with a nuclear export receptor (exportin 1) which then allows the RNA transcript to leave the nucleus despite haveing introns in the sequence
Eukaryotic initiation factors 2, 2B, 3, 4A, 4E, 4G, and PABP’s impact on translation
eIFs:
eIF2 - binds tRNAᵢᴹᵉᵗ to the ribosome using GTP
eIF2B - a heteromeric guanine nucleotide exchange factor that separates GDP and eIF2 (a GTPase)
eIF3 - scaffold protein (binds and interacts with many other initiation factors)
eIF4A - has RNA helicase and ATPase activity and helps bind mRNA to the ribosome
eIF4E - binds to the mRNA cap and gives the ribosome a place to bind
eIF4G - binds to mRNA molecules and supplies other eIFs (including eIF4E) with a place to bind
PABP:
As well as protecting the Poly-A tail from nuclease degradation, it acts as an RNA-binding protein, triggering the binding of eukaryotic initiation factor 4 complex (eIF4G) directly to mRNAs Poly-A tail
Translation in eukaryotes: the initiation
The initiating tRNA (containing methionine) binds to the small subunit of the ribosome (it is the only tRNA capable of binding to the small subunit without the whole ribosome clamped together) with help from eukaryotic initiation factor 2 (eIF2) which acts as a GTPase, hydrolysing GTP as an energy source for this process.
The ribosome then binds to the 5’ end of the mRNA due to help from eIFs (eIF4E, eIF4G, etc) and it then searches the mRNA for the start codon (AUG), with support from eIF4A acting as a helicase to move through RNA’s secondary structure
Once the start codon has been found, the eIFs dissociate and translation elongation occurs
What is ‘leaky scanning’ and how can it be used effectively?
The process of ignoring certain AUG codons based on a nucleotide not matching the consensus nucleotide sequence
This can be used to generate the same proteins with different N-terminals which can act as differences in signalling, instructing proteins to move to different locations, while still retaining the same function
