Lecture 6 - Transcription in Prokaryotes Flashcards
what is mRNA?
mRNA or messenger RNA are used for encoding the amino acid sequences for polypeptides
what is tRNA?
tRNA is transfer RNA which is used for matching amino acids to the mRNA during protein synthesis
what is rRNA?
rRNA is ribosomal RNA which is used to make ribosomes with catalytic properties
what is miRNA?
miRNA is micro RNA which is used for regulation of genes
what is the role of RNA in transcription and protein synthesis?
RNA is made using DNA as a template. transcription is regulated to control amount of protein
Why is RNA able to catalyze reactions?
RNA can be compact since it is single stranded and uses ions for reactions
what kind of processing occurs in eukary RNA?
elimination introns and joining of exons, adenylation of 3’ end, capping at the 5’ end
how is RNA synthesis similar to DNA synthesis?
Nucleophilic attack of the 3’ OH group on the a-phosphoryl group of the rNTP; the rxn is driven by elimination of an inorganic phosphate and its hydrolysis
what role does DNA play in RNA synthesis reaction?
provides the template (which nucleotides are added) and promoter which defines which sequences are transcribed
what are the differences in RNA and DNA synthesis?
- the use of rNTP or ribonucleotide triphosphates instead of dNTPs
- the use of uracil instead of thymine;
- RNA synthesis requires a DNA template and promoter sites,
- RNA synthesis does not require a primer
- one strand is made
- many errors are made since there isn’t proofreading
why does it not matter if RNA is error prone?
mRNAs are small so chances that there is error is small; most RNAs are made to last a short time and a bunch are made at a time; mRNA is made to be disposable
what makes RNA so unstable?
RNA contains ribose instead of deoxyribose. It has a 2’ OH group competing with the 3’ OH group for the phosphodiester bond through the neighbor group effect. RNA is easily changed at slightly akali pH whereas the diester bond in deoxyribose is relatively stable.
What is the mechanism of how RNA is unstable in an alkali environment?
-OH attacks the 3’ OH and that attacks the phosphodiester bond, the result is a 2’,3’ cyclic monophosphate derivative and a shortened RNA
describe RNA Polymerase in prokary
bacteria only have 1; RNA Pol has 5 core subunits alpha-2-beta-beta’-omega and a 6th called sigma; it does not have a 3’ to 5’ exonuclease so high error rate; it binds to promoter regions to start transcription
What is RNA Polymerase made of?
it has 2 alpha subunits responsible for assembly and attaching to upstream promoter sequences; it has beta and beta’ subunits that are catalytic units and responsible for DNA binding; the sigma portion guides it to the promoter; the omega part protects the enzyme from denaturation
where does the sigma part of RNA polymerase bind to?
it binds to the -10 and -35 TATA sequences
where does the alpha part of RNA polymerase bind to?
it binds to the A-T rich upstream promoter element that is between -40 and -60. these promote strand separation. These sequences determine the efficacy of RNA Pol binding = affect gene expression
how to identify promoter sequences?
called footprinting. you have identical DNA fragments that are tagged radioactively. you add DNAse to both the control and the one with the RNA Pol. cuts are not made where RNA Pol is bound so you will get different lengths of DNA. Use of gel electrophoresis to figure it out. missing bands = RNA Pol binding
what is the idea behind footprinting?
DNA that is bound by RNA Pol would not be cut by the DNAse
What is the mechanism for RNA polymerase reaction?
reaction involves 2 Mg2+ ions and 3 Asp residues; Mg2+ facilitates attack by the 3-OH group onto the nucleoside triphosphate and helps displace the pyrophosphate
