RNA 8.23 Flashcards
what are the four main categories of RNA and their functions?
messenger RNA (mRNA) - encode amino acid sequences - 5% of total RNA Transfer RNA (tRNA) - match amino acids to triplet codons - 15% of RNA Ribosomal RNA (rRNA) - catalyze the formation of polypeptides - 80% of RNA Micro RNA (miRNA) - regulate expression of genes
What are ribozymes
RNA molecules that act as catalysts with metal ion cofactor
explain how pre-mRNA is manufactured (eukaryotes only)- include the stage names
5’->3’ direction
Initiation - this is the rate limiting step - RNA polyermase core binds the DNA promoter, helicase unwinds the DNA and a transcription bubble forms
Elongation - sigma subunit dissociates and is replaced by NusA - RNA polymerase adds dNTP’s onto the growing strand via a nucleophilic attack of the alpha phosphate on the 3’-OH group - as the DNA unwinds, topoisomerases relieve supercoiling stress
Termination -
p-independent - GC-rich region signals the end of the DNA to be transcribed - a hairpin turn of the RNA is formed which disrupts attachment to the template DNA - the AT-rich region after GC is unstable and causes RNA to be released - NusA dissociates and RNA polymerase releases from the template strand
protein-dependent - the transcribed RNA will contain a CA-rich sequence known as a rut (Rho utilization element) site - this recruits a p (rho) protein which travels along the RNA 5’->3’ until it encounters RNA polymerase - p-protein then separates the RNA from the polymerase complex
give the basic characteristics of RNA polymerase in prokaryotes
a single RNA polymerase makes mRNA, rRNA, and tRNA
5 core subunits + 1 sigma subunit = holoenzyme
Core enzyme polymerizes the RNA, sigma subunit allows the core to recognize the promoter
RNA polymerase lacks 3’->5’ exonuclease function and thus cannot proofread in that direction - leads to greater error rate
RNA polymerase binds to promoter regions to initiate transcription
Describe the characteristics of a stretch of DNA that RNA will be coded from
promoter - RNA polymerase will bind here
operator - binding site for repressor of the activator
structural gene - DNA containing the target gene (may contain many other genes)
together, these are referred to as the operon
Names for the three strands in RNA transcription
Template strand
Coding strand - the non-template DNA, has the same sequence as the RNA (except for Uracil)
RNA
Describe specificity in bacterial RNA transcription
different sigma factors recognize different promoter sequences, thus, they specify which genes will be expressed (decreases affinity but increases specificity)
the consensus sequence is the most readily recognizable promoter sequence by sigma factors - strong promoters will conform closely to the consensus sequence
Describe the 3 different eukaryotic RNA polymerases
Describe the 3 different eukaryotic RNA polymerases
RNA polymerase I - makes pre-rRNA (the precursor to 28S, 18S, 5.8S)
RNA polymerase II - makes mRNA
RNA polymerase III - makes tRNA and other small RNA products
what are certain targets of antibiotics in regards to RNA transcription
inhibit RNA polymerase, intercalating agents, chain terminators that lack 3’OH (similar to what a ddNTP)
how is DNA in prokaryotes and eukaryotes different in regards to RNA transcription
eukaryotic DNA contains coding (exon) and noncoding (intron) regions
Describe the 5’ cap and 3’ tail
5’ cap protects the RNA from nucleases and improves translation efficiency
7-methylguanosine is added to the 5’ end of RNA as it appears from the polymerase - 5’-5’ triphosphate linkage
3’ tail or Poly-A tail - protects RNA from nucleases and improves translation efficiency - RNA contains a polyadenylation signal followed by an untranslated region of RNA - an endonuclease will cut the RNA after its polyadenylation signal and a polymerase will add on a poly(A) tail
explain the process of splicing
a pre-mRNA strand will contain coding (exon) and noncoding (intron) regions
oftentimes the large majority of the pre-mRNA will consist of introns
introns are removed by splicesomes - the start and ends of introns are bordered by consensus sites that splicesomes recognize - the 5’ end is the donor site and the 3’ end is the acceptor site
a branch site in the middle of the intron will form a phosphodiester bond with the 5’ donor site
the freed exon 3’ will then nucleophilically attack the intron 3’ acceptor site (thus linking the exons together)
what is the role of snRNP in RNA splicing
used for splice site recognition - U1 binds the 5’ donor site - U2 binds the branch site
U4, 5, and 6 come together with U1 and U2 to form a splicesome
explain the principle of alternative splicing
removing certain exons along with introns allows the formation of several different types of mRNA from one variant of pre-mRNA
What is Marfan syndrome? cause?
a splicing disease characterized by aortic aneurysm, arachnodactyly, and dural ectasia (enlargement of the dura around the brain and spinal cord)
present theory is that the gene for fibrillin (a form of connective tissue) is improperly spliced - leads to two things
- inferior connective tissue (explains aortic aneurysm and dural ectasia)
- a homolog for fibrillin is a protein that suppresses TGF-B (growth factor), fibrillin can als supress TGF-B - however defective fibrillin cannot - TGF-B overexpression leads to arachnodactyly
What role does lactose play in a bacteria
when glucose levels are low, bacteria use B-galactosidase to convert lactose into galactose and glucose for more metabolic fuel
what are the three factors needed for regulational control at the transcriptional level
regulatory gene that codes for a repressor or activator
promoter sequence that can be bound by RNA polymerase
operator sequence that can be bound by a repressor
what is the function of an inducer
inducers bind to repressors to weaken affinity for the operator
negative regulation vs positive regulation at the transcription level
negative regulation involves action from a repressor potein - either the repressor is knocked off of DNA or is induced to bind
positive regulation involves use of an activator - either the activator is knocked off of DNA or is induced to bind
how is B-galactosidase expression upregulated?
low glucose = high cAMP
cAMP binds to catabolite activator (CAP) - heterodimer then binds near the promoter which then facilitates RNA polymerase binding to promoter
how does bacteria avoid making B-galactosidase when lactose is not present? how does the presence of lactose change this
repressor is consistently bound to the operator
when lactose is present and metabolized, allolactose (an inducer) binds to the repressor and inactivates it
Describe B-galactosidase activity with differing glucose and lactose levels
High glucose - very little cAMP present - no way to form the activating heterodimer - no B-galactosidase activity
Some glucose - still only a little cAMP present
low glucose/high lactose - allolactose (inducer) inactivates the repressor (causing it to fall off of the operator) - high cAMP binds to CAP - heterodimer binds near promoter and recruits polymerase to bind promoter
what is the difference between prokaryotic and eukaryotic regulation
prokaryotic regulation is only at the transcriptional level - all mRNA is going to be translated into protein
eukaryotic regulation is controlled at many levels including transcription and translation - chromatin structure (loops, condensed structure, etc) also contributes to regulation
What are the five types of proteins required for eukaryotic RNA polymerase activity
transcription activators, coactivators, basal transcription factors, architectural regulators (DNA looping), chromatin modification/remodeling proteins
describe the transcription process in eukaryotes
TATA binding protein (TBP) binds the TATA box in the promoter region of the operon
other proteins (including polymerase) are recruited and form the preinitiation complex (pic) - this is the rate limiting step
transcription bubble forms and transcription is initiated - phosphorylation of the carboxyl-terminal domain of RNA polymerase allows it to move past the promoter region - elongation is assisted by elongation factors
once termination point is reached, elongation factors dissipate, RNA is replease, and the transcription complex dissociates
how do repressors and activators work in eukaryotic transcription
activators attach to enhancer sequences upstream from the target gene - mediators attach to the activators and recruit TBP to bind the TATA box which then recruits the rest of the PIC
repressors work by stopping mediators from binding to the activators
What functions can transcription factors serve? How does this increase the versatility of regulation in eukaryotes
Functions - DNA binding, factor dimerization, transcriptional activation
Different transcription factors have different mixes of domains which serve these functions - level of expression of these transcription factors allows for very nuanced regulation - to make these more specific, many of the factors that interact with DNA are sequence specific
note: DNA binding proteins often look for a binding site consisting of palindromic sequences
