Rod Devenish; Week 3 MED1011; Biochemistry
RNA polymerase is the enzyme that uses DNA as a template to make RNA. Initiation of transcription requires recognition and binding of a promoter sequence on DNA, promoter indicates template. Binding can be influenced by other proteins. After binding, RNA polymerase unwinds the DNA about 10 base pairs at a time and reads the template in the 3' to 5' direction. The RNA is synthesised in the 5' to 3' direction. As RNA is formed it peels away, allowing DNA rewinding. Energy for synthesis comes from removal of pyrophosphate group from each nucleoside triphosphate (NTP). Particular base sequences specify termination. In eukaryotes, this involves a protein complex and does not occur until a sequence signalling eventual polyadenylation has been transcribed.
mRNA codes for a protein, rRNA contributes to structure and function of ribosomes, tRNA carries an amino acid and is an adaptor molecule allowing decoding of RNA to protein (transfer RNA). Prokaryotes have one kind of RNA polymerase for all three, but eukaryotes have 3 (I, II and III). RNA polymerase II makes all mRNA in eukaryotes. RNA is not proof read and mistakes occur every 10^4 to 10^5 bases incorporated.
Codons are groups of 3 nucleotides. There are 64 possible codons. mRNA is read in codons. They code for only 20 amino acids and all start and stop signals. Many codons are therefore redundant, but the code is not ambiguous. It is also near universal, with minor variations in mitochondria and chloroplasts.
- Stop: UAA, UAG, UGA; Start: AUG (methionine)
mRNA is processed in eukaryotes before it leaves the nucleus. A methyl-G cap is added at the 5 end, and a poly-A tail at the 3 end following cleavage at the AAUAAA sequence. These are important for stability.
Introns are removed using spliceososme, a complex of RNA and proteins. As soon as pre-mRNA (before modification) is transcribed several snRNPs are added (small nuclear ribonucleoproteins) which links the coding regions as a single sequence. Spliceosomes are formed by snRNP.
Different places DNA can be modified are at histones, being transcribed, mRNA being processed, transport control leaving the nucleus, mRNA stability control to stop it becoming inactive, translational control of protein synthesis, posttranslational control of protein activity or protein degradation.
Major method of transcriptional control is selective transcription dictated by transcription factors binding to regulatory sequences of DNA. Specific transcription factors must bind to the promoter before RNA polymerase can bind. Some promotion factors are 'general' and these work with RNA polymerase II to transcribe mRNAs. Initiation also depends on activator and repressor proteins bound.
The first transcription factor, TFIID, binds to the promoter after the TATA box and another transcription factor (B) joins it. RNA polymerase II binds only after several transcription factors are already bound to DNA. More transcription factors are added and the RNA polymerase is ready to transcribe DNA.
A long stretch of DNA can lie between an activator binding site and the transcription complex. DNA can fold to bring the activation site with an activator protein into contact with the transcription complex.
The simultaneous control of widely separated genes can be possible through proteins that bind to common sequences in their promoters (translation of one regulatory protein that promotes 3 genes).
Alternative splicing of genes containing several exons can be used to produce different proteins.
The stability of mRNA in the cytoplasm can be regulated by protein binding. Specific AU rich sequences mark some mRNA for rapid breakdown by a ribonuclease complex (exosome).
One class of RNA that regulates gene expression are the microRNAs (miRNA), which are short, 19-25 nucleotide, non-coding RNA. They bind some complementary sequences in other RNA and prevent translation and facilitate degradation. They are cut into these small fragments by dicer protein, and another protein converts them to single stranded RNA. miRNAs may regulate 10-30% of human genes, each miRNA may regulate 200 target genes (there are about 500-1000 miRNA genes in the human genome). Many genes have target sites for a few miRNAs.
miRNA tends to be de-regulated in some malignancies, miR-17-92 miRNA cluster on chromosome 13 is often increased in B cell lymphomas. Elevated levels of these miRNAs are found in clinical samples and related cell lines. They have a potential for diagnostic use.
mRNA is not directly linked to the amount of protein produced, concentration is determined after strands are produced, probably by suppressor proteins that bind to some mRNA to inhibit translation.
Proteins are usually modified after translation, regulating the lifetime of a protein is a way to control its actions. Proteins identified for breakdown are often linked to a 76 amino acid protein, ubiquitin. Other ubiquitin chains attach to the primary one. The protein-ubiquitin complex attaches to a proteasome, inside which energy from ATP cuts off the ubiquitin (recycled) and unfold its target protein. Proteases digest the protein into small peptides and amino acids.