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
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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
Which two regions does sigma 70 recognize with consensus sequences?
-10 and -35; if there are different sigma factors then it will recognize different sites; most common is sigma 70
On the growing RNA strand, where is the nucleoside triphosphate added?
the 3’ end of the RNA strand
is the RNA strand created complementary or not to the DNA strand?
it’s complementary
What catalyzes RNA transcription and how long can the enzyme go for?
RNA polylmerase; it can go on for 35bp on DNA
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
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what is the overview of transcription in prokary?
- RNA Pol binds to promoter
- growing end of RNA temporarily base pairs with DNA strand about 8bp
- DNA duplex unwinds and creates bubble of 17bp
- this creates positive supercoils which is relieved by topoisomerases
what is the DNA template strand?
DNA template strands serves as template for RNA polymerase
what is the DNA coding strand?
DNA coding strand is the non template strand but has the same code as the RNA transcript; also has the regulatory sequences
what happens in initiation of transcription in prokary?
- RNA Pol binds to promoter to create closed complex where DNA is not unwound
- Open complex forms from -10 to +2; the region unwinds to free up template strand
- some NTPs are added but are wasted until it achieves at least 10 nucleotides
- promoter clearance! ternary complex formed
what happens in elongation in transcription in prokary?
- once the RNA pol escapes promoter elongation proceeds
- DNA is separated and goes different paths
- one nucleotide is added at a time at 50-90 NT/sec
- Proofreading can occur
what is kinetic proofreading?
RNA pol slows when it is on top of a mismatch and uses pyrophosphorolysis to get back an rNTP
what is nucleolytic proofreading?
RNA nuclease activity can remove a few NTs and eliminate them as NMPs
what happens during prokary termination of transcription?
RNA pol can stop due to certain sequences on the DNA. it can also stop thru interaction with NusA
what is rho-independent termination?
there are 3 U’s near the 3’ end of the transcript which forms a 15-20NT hairpin that causes the RNA Pol to fall off the DNA due to sterics
what is rho dependent termination?
CA rich sequence called rut (rho utilization element); rho protein processes till termination then rho protein binds to rut site and migrates along the mRNA in a 5’ to 3’ and acting as a helicase that separates the mRNA from the DNA template
what are some differences from eukary and prokary transcription?
eukary RNA undergo processing (poly AAAA, 5’ cap, and introns and exons) while in prokary, translation can happen during transcription which can also speed transcription up
why is transcription regulated?
it requires a lot of energy
how is transcription regulated?
- regulate RNA Pol affinity for promoter through
- promoter sequence
- activator proteins
- repressor proteins
how do the transcription regulators interact with the DNA?
they interact by
what is an operon?
set of genes that is coded into single mRNA, common in bacteria but not eukary, genes in a metabolic pathway
what is an operator?
sequence of DNA about 15NTs that is recognized by a gene regulatory protein; Trp repressor binding site
what is the Trp operon?
makes Trp; regulated by Trp; continuously transcribed
what is a constitutive gene?
a gene that continuously transcribes
what is the Trp repressor protein?
binds to the Trp operon; prevents transcription of mRNA, allosteric protein that increases affinity for DNA when Trp is present = high Trp = greater affinity
how is gene expression controlled by activator proteins?
genes are usually expressed at a low level; CAP has to bind to cAMP then binds to DNA; it facilitates transcription of DNA
what is the lac operon? How is it regulated?
makes lactase and associated proteins; involves a lac repressor and activator protein CAP; regulated by levels of lactose and glucose; operon is expressed when activator is on and repressor is not bound
what are the parts of the lac operon?
lacI codes for repressor, lacY codes for galactoside permease, lacZ codes for beta galactosidase which breaks down lactose into glucose and galactose; lacA codes for unknown
how is lactose used by e. coli?
lactose is not preferred energy source so it will use all glucose before turning on lac operon
how does glucose, CAP, cAMP regulate the lac operon?
glucose regulates expression of lac operon thru CAP; levels of glucose and cAMP are inversely correlated, CAP-cAMP needs to present for lac operon to express
how does lactose regulate the lac operon?
lacI is always expressed (constitutive) so repressor is always on and lac operon is turned off, but when lactose is present, allolactose binds to repressor and that alters the shape of it and it allows the lac operon to express