Last updated on January 11, 2020 at 14:29
- RNA uses uracil instead of thymine, and riboses instead of deoxyriboses, like DNA does
- RNA is transcribed by RNA polymerase, which requires Asp and Mg2+, just like DNA polymerase
- There are three types of RNA, ribosomal, transfer, and messenger RNA
- Ribosomal RNAs are a component of ribosomes
- Transfer RNAs are essential for translation
- It’s cut at both ends by RNase P and RNase D
- a CCA sequence is added to the 3′ end of the tRNA
- the CCA sequence is where the amino acid will bind to
- Methylation, deamination, reduction of bases
- Messenger RNA is the RNA that is used as a template for translation into protein
- Immature mRNA is processed by capping, tailing, splicing and cleavage before it’s mature
- Capping involves 7-methylguanosine cap to the 5′ end
- Tailing involves attaching a poly(A) tail on the end of the mRNA
- By the enzyme poly-A polymerase
- Splicing involves excising the introns in the mRNA and linking the exons together
- Transcription and translation happens at the same time and place in bacteria, but not in eukaryotes
Transcription in bacteria
The RNA molecules don’t differ much from DNA. It uses ribonucleotides instead of deoxyribonucleotides (which differ by just a hydroxyl group), and it uses uracil instead of thymine. However, these differences are enough to create large variations in structure between the two. RNA molecules can create structures like hairpins, loops and bulges, which gives RNA the possibility to not just be a double helix like DNA, but other shapes.
RNA polymerase, like DNA polymerase, needs aspartate and Mg2+ to work. Other similarities between the two are: both synthesize in 5’ -> 3’ direction, they have the same mechanism of elongation and they have similar processivity. RNA pol is different from DNA pol by how it doesn’t need a primer and has no nuclease activity, and that it always creates a cap on the 5’ end of the molecule.
When RNA polymerase transcribes DNA, it must first unwind a portion of the coiled DNA into two separate strands, the nontemplate strand (which the new RNA will be equal to) and the template strand, which RNA pol will use as a template. The unwound portion is called the “transcription bubble”. When RNA pol works its way along the DNA molecule, it will unwind new DNA and rewind the DNA it has already copied. In this way, the transcription bubble is always moving.
RNA polymerase has five different subunits. They are the α, β, β’, σ and ω subunits. The σ subunit recognizes promoters and can be switched out with different σ subunits. Each σ subunit recognizes specific promoters for specific genes. There exist several types of σ subunits. The “standard” σ subunit is called σ70, which recognizes and binds “housekeeping” genes, genes that should be expressed when nothing special is happening in the cell. However, if the cell is exposed to some sort of stress, for example an increase in temperature, it will switch out the σ70 subunit for a different one, in this case σ32, which recognizes promotors for so called “heat shock genes”, genes that should be expressed in response to stress. By switching out the σ subunit, the cell can rapidly change what kinds of genes it wants transcribed. Without the σ subunit, RNA polymerase cannot bind a promoter, and therefore cannot transcribe anything.
We’ve looked at how RNA polymerase begins transcription, but how does it know when to stop transcribing? It can be done in two ways; by the function of a protein called rho-protein (rho-dependent termination) or without it (rho-protein independent termination).
In rho-dependent termination, a protein called rho-protein, which is a helicase, binds to the “tail” of the RNA polymerase when it begins transcription. Rho travels up this tail towards the “body” of RNA pol, and when it reaches it, it will separate the RNA from the RNA polymerase enzyme.
In rho-independent termination, the transcribed RNA molecule creates a hairpin loop on itself, on the end. This hairpin loop on the end of RNA causes the RNA to dissociate from RNA polymerase.
Several medications and toxins are RNA polymerase inhibitors. The ones you should know are in the table below.
|Name of compound||Actinomycin D||Rifampicin||α-amanitin|
|Inhibits which RNA polymerase?||All||RNA polymerase||II and III|
|In bacteria or eukaryotes?||Both||Bacteria||Eukaryotes|
|Mechanism of action?||Binds to DNA, blocking RNA polymerase||Binds to beta subunit, blocking initiation of transcription||Blocks mRNA synthesis by binding to RNA pol.|
Transcription in eukaryotes
Transcription in eukaryotes is very similar to in prokaryotes, however, there are differences.
Prokaryotes don’t have nuclei in their cells. This means that DNA and the ribosomes are located at the same place (in the cytosol). Eukaryotes however, have their DNA in the nucleus, separated from the ribosomes. Because transcription must happen where the DNA is, and translation must happen where the ribosomes are, the first difference becomes clear. In prokaryotes, RNA is transcribed and translated at the same time at the same place, while in eukaryotes, RNA is first transcribed in the nucleus, then transported out of the nucleus into the cytoplasm, where it is translated into protein.
Bacteria only have one RNA polymerase, but eukaryotes have three. RNA polymerase I, which is located in the nucleolus and transcribes rRNA, RNA polymerase II, which is located in the nucleus and transcribes mRNA and snRNA, and RNA polymerase III, which is also in the nucleus, but transcribes tRNA and rRNA.
RNA is processed in many ways before it is translated into protein. The most important are capping, tailing, splicing and cleavage. We call transcribed mRNA immature until it is processed in these manners. At that time, it becomes mature mRNA, ready for translation.
All eukaryotic mRNA has a 5’ cap and a poly(A) tail on the 3’-end. The cap is composed of a methylated adenine or guanine and three phosphate groups. The cap protects the mRNA, and facilitates binding to the ribosome.
The poly(A) tail is composed of 100-250 adenine residues on the 3’-end of the mRNA. Its function is protection and transport. The tail is created by poly-A polymerase. The reaction is like this (and yes you need to know this):
RNA-nATP —-> RNA-(AMP)n+nPPi
Immature mRNA is composed by introns and exons. Introns are sequences that are not meant for translation, so they are “spliced out” (removed). They can be spliced out by enzymes called the spliceosome, or more precisely small nuclear ribonucleoprotein particles (snRNPs), or they can be spliced out by the enzymatic activity of the mRNA itself. The last way is called “self-splicing”.
Alternative splicing can yield completely different products, very similar products, or anything in between. This works by splicing mRNAs differently. Consider the figure above. If every intron except intron “G” was spliced out, the mature mRNA, and the protein it codes for, would be different. By varying what we splice out and what we leave, we can change the resulting protein completely.
tRNAs are processed in a certain way. After transcription (by RNA pol III), they are cut at both the 5’-end and the 3’-end by RNase P and RNase D respectively. The sequence CCA is added to the 3’-end, and many of the tRNA bases are modified by methylation, deamination and reduction. CCA is important, as it’s where the amino acid will bind to.
Base modification doesn’t just happen at tRNAs, however. mRNA bases can also be modified. For example, deamination of a cytosine residue will turn it into a uracil.
20. DNA repair
22. Short RNAs