RNA 8.23

Question 1

what are the four main categories of RNA and their functions?

Answer 1

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

Question 2

What are ribozymes

Answer 2

RNA molecules that act as catalysts with metal ion cofactor

Question 3

explain how pre-mRNA is manufactured (eukaryotes only)- include the stage names

Answer 3

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

Question 4

give the basic characteristics of RNA polymerase in prokaryotes

Answer 4

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

Question 5

Describe the characteristics of a stretch of DNA that RNA will be coded from

Answer 5

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

Question 6

Names for the three strands in RNA transcription

Answer 6

Template strand

Coding strand - the non-template DNA, has the same sequence as the RNA (except for Uracil)

RNA

Question 7

Describe specificity in bacterial RNA transcription

Answer 7

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

Question 8

Describe the 3 different eukaryotic RNA polymerases

Question 9

Describe the 3 different eukaryotic RNA polymerases

Answer 9

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

Question 10

what are certain targets of antibiotics in regards to RNA transcription

Answer 10

inhibit RNA polymerase, intercalating agents, chain terminators that lack 3’OH (similar to what a ddNTP)

Question 11

how is DNA in prokaryotes and eukaryotes different in regards to RNA transcription

Answer 11

eukaryotic DNA contains coding (exon) and noncoding (intron) regions

Question 12

Describe the 5’ cap and 3’ tail

Answer 12

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

Question 13

explain the process of splicing

Answer 13

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)

Question 14

what is the role of snRNP in RNA splicing

Answer 14

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

Question 15

explain the principle of alternative splicing

Answer 15

removing certain exons along with introns allows the formation of several different types of mRNA from one variant of pre-mRNA

Question 16

What is Marfan syndrome? cause?

Answer 16

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

1. inferior connective tissue (explains aortic aneurysm and dural ectasia)
2. 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

Question 17

What role does lactose play in a bacteria

Answer 17

when glucose levels are low, bacteria use B-galactosidase to convert lactose into galactose and glucose for more metabolic fuel

Question 18

what are the three factors needed for regulational control at the transcriptional level

Answer 18

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

Question 19

what is the function of an inducer

Answer 19

inducers bind to repressors to weaken affinity for the operator

Question 20

negative regulation vs positive regulation at the transcription level

Answer 20

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

Question 21

how is B-galactosidase expression upregulated?

Answer 21

low glucose = high cAMP
cAMP binds to catabolite activator (CAP) - heterodimer then binds near the promoter which then facilitates RNA polymerase binding to promoter

Question 22

how does bacteria avoid making B-galactosidase when lactose is not present? how does the presence of lactose change this

Answer 22

repressor is consistently bound to the operator

when lactose is present and metabolized, allolactose (an inducer) binds to the repressor and inactivates it

Question 23

Describe B-galactosidase activity with differing glucose and lactose levels

Answer 23

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

Question 24

what is the difference between prokaryotic and eukaryotic regulation

Answer 24

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

Question 25

What are the five types of proteins required for eukaryotic RNA polymerase activity

Answer 25

transcription activators, coactivators, basal transcription factors, architectural regulators (DNA looping), chromatin modification/remodeling proteins

Question 26

describe the transcription process in eukaryotes

Answer 26

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

Question 27

how do repressors and activators work in eukaryotic transcription

Answer 27

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

Question 28

What functions can transcription factors serve? How does this increase the versatility of regulation in eukaryotes

Answer 28

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

Question 29

note: DNA binding proteins often look for a binding site consisting of palindromic sequences