Module 4 Sections 1-3 Flashcards
Transcription, mRNA Processing, Genetic Code
list all tRNAs, rRNAs, snRNAs, snoRNAs
snRNAs = small nuclear RNAs, play a role in gene regulation - form part of the spliceosome that is important for mRNA processing
mRNA = only type of coding RNA – determines the amino acid sequence of a protein
rRNA = ribosomal, present during translation
tRNA = transfer, present during translation
snoRNA = small nuclear RNA, involved in the processing of rRNAs
miRNA – micro RNAs, limit translation by binding to 3’-end of target mRNAs
IncRNA = long non-coding RNAs, can be important regulatory RNAs
transcription vs DNA replication characteristics
uses template strand: both
has initiation, elongation and termination: both
proceeds in the 5’-3’ direction: both
requires primers for initiation: DNA replication
has specific start and stop sites: both
is selective: transcription
rNTP’s are used as building blocks: transcription
both strands are used simultaneously as a template: DNA replication
the template strand
RNA polymerase uses 3’-5’ template DNA strand to make RNA - RNA will be complementary to the template strand but MATCH the sequence of the coding strand, replacing T with U
transcriptional start site
Where RNA polymerase starts reading
Distinct from translational start site
Which encodes the first amino acid of the protein
translational start site
Generally AUG (met)
Located many nucleotides upstream the initiation codon
Region between TSS and start codon = the 5’ untranslated region (5’UTR)
bacterial RNA polymerase holoenzyme subunits
5 polypeptide subunit core with 3 subunits (B, B’, w) and 2 alpha subunits
sigma factor
acts as a transcriptional initiating factor and adds DNA binding selectivity
main mechanic of RNA synthesis
3 aspartic acid residues in the RNA pol active site, which capture and coordinate two Mg2+ ions
Mg2+ ions role in RNA synthesis
1 of them interacts with the phosphate groups of the NTP and the other acts to bring the 3’OH of the last added nucleotide in close enough proximity to the incoming rNTP of a nucleophilic attack reaction to take place on the alpha phosphate, releasing PPi
does RNA polyermase have a built-in proofreading center
no
RNA polymerase alternative proof-reading mechanisms
- kinetic proofreading
- nucleolytic proofreading
kinetic proofreading
If an incorrect nucleotide is added, the proper H-bonding isn’t formed, which causes fraying at the DNA-RNA duplex
When recognized by the RNA pol, it can stall until pyrophosphorolysis reverses the reaction at this base pair, allowing for the correct rNTP to be paired
nucleolytic proofreading
RNA polymerase must backtrack to the mismatched base to fix the error
Must reverse its direction by a few nucleotides in the template and uses its intrinsic endonuclease activity to hydrolyze the phosphodiester backbone of the transcript upstream of the error, removing the incorrect base
kinetic vs nucleolytic proofreading
kinetic = the polymerase stalls, allowing pyrophosphorolysis to remove the incorrect base
nucleolytic = the polymerase backtracks and hydrolyzes the phosphodiester bond upstream of the incorrect base
phases of transcription
- initiation
Occurs as RNA polymerase binds to specific DNA sequences called promoters
These sequences contain specific elements and are located upstream of the TSS
- elongation
the process of adding nucleotides to the growing RNA strand - termination
the release oft he product RNA when the polymerase reaches the end of a gene or other transcription unit
5 main steps in the process of transcription
- The polymerase binds the promoter, assisted by sigma factors in prokaryotes or transcription factors in eukaryotes
- Open complex is formed, in which bound DNA is partially unwound near a region of 10bp upstream of (ahead of) the transcription start site
- Transcription is initiated within the complex, leading to a conformational change that converts the complex to the form required for elongation
- Promoter clearance, involving movement of the transcription complex down the DNA template and away from the promoter, leads to the formation of a tightly bound elongation complex
- Once elongation begins, RNA polymerase becomes a highly efficient enzyme, completing synthesis of the transcript before dissociating from the DNA template, and then recycling for a new round of transcription
sigma factors in transcriptional initiation
a transient subunit of the bacterial RNA polymerase that directs the enzyme to the promoter - different sigma factors are specific for different promoters
most common sigma factor
O^70, 70kDa as molecular weight
consensus sequence
a sequence of nucleotides or amino acids that has a similar structure and function in different organisms
-35 and -10 regions
important interaction sites for O^70
-10 region: 5’-TATAAT-3’
-35 region: 5’-TTGACA-3’
upstream promoter
AT-rich recognition element between -40 and -60 in the promoters of highly expressed genes bound by one of the subunits of RNA polymerase
importance of consensus sequences, UP sequences, and spacer
The efficiency with which RNA polymerase binds to a promoter and initiates transcription is determined by the –10, -35 and UP sequences (and the spacer), and the distance of the UP element from the transcription start site
spacer length importance
any changes to the spacer length (larger or smaller) will reduce transcription
2 turns of a double helix allows the orientation of consensus sites to be on the same side of the helix
increase in spacer length
longer than 2 turns of a double helix = consensus sites are no longer on the same side of the helix = transcription is reduced
steps in bacterial transcription
- initiate bacterial transcription
- Sigma factor must bind to the promoter region, contacting the –35 and –10 sites
- The enzymes is in the closed conformation
- N-terminus of the sigma factor is blocking the DNA entry channel - open conformation
- Pincers of the Pol enzyme closes around DNA, and the N-terminus moving from the active site cleft
- Independent of ATP for all sigma factors except O&54, which is ATP-dependent - abortive initiation
- RNA polymerase does not require a primer
- Must bind and hold 2 nucleotides in place for long enough to catalyze the phosphodiester bond
- After this bond and for the first 8-10 bonds, there is a high chance that the polymerase will release the transcript without extending it further
- Energetically favourable for the RNA polymerase to release the 8-10 base pair RNA transcript from the initiation complex
- Holoenzyme will begin NA synthesis again on the same template - elongation
- RNA polymerase can hold onto the transcript long enough to extend it beyond 10 nucleotides
- At this point, the RNA becomes stable and clears the promoter to enter the elongation phase
- RNA polymerase will continue along the DNA template until it reaches a termination signal
- Elongation allows for the promoter sequence to clear from the polymerase
- Weakens the hold of the sigma factor, which falls off the polymerase - termination
- Enzyme releases the DNA template only when it encounters a termination sequences
- Polymerase is processive, moving smoothly along the template, synthesizing the complementary RNA strand and dissociating only when the transcript is complete
open vs closed conformation
closed: bound DNA is intact
open: bound DNA is partially unwound upstream of the transcription start site
RNA polyermases in eukaryotic cells
- RNA Pol I
- Ribosomal RNA (rRNA) precursors
- Responsible for transcribing rRNAs
- 80% of all transcription - RNA Pol II
- Messenger RNA (protein coding genes)
- Responsible for the synthesis of mRNA - RNA Pol III
- Smaller functional RNAs (tRNAs, snRNAs)
how many subunits does RNA Pol have for eukaryotes
12
other uses for transcription inhibitors
some species rely on transcription inhibitors for natural biodefense
Ex: mushroom Amanita phalloides (the death cap) produces a-amanitin, a cyclic peptide that disrupts eukaryotic mRNA synthesis by blocking Pol II
general structure of eukaryotic promoter
Core promoter that consists of a region that is recognized by the general transcription factors which are able to recruit RNA Polymerase
Has both upstream and downstream regulatory sequences
Bind gene-specific transcription factors that can act as activators (or repressors) of transcription at that locus
differentiation
the process where a cell changes from one cell type to another
transcription factors involved in the differentiation of cells
- developing organisms starting out in a pluripotent state and move to a a higher state like a blood cell, brain cell, pancreatic cell, etc
- they turn on specific subsets of genes to drive them towards their terminal fate (committed to a particular function, can no longer divide)
- done by genes that regulate the transcription of other genes by binding to promoters and interacting with the transcriptional machinery - using skin fibroplast to change a cell’s gene expression
- involves induced pluripotency by pushing cell back to its embryonic stem cell state - cellular reprogramming
- using genetic engineering to force the fribroblast t oexpress the TFs or providing the fibroblasts with the actual TF protein
pluripotent state
stem cells that can differentiate into cells derived from any of the 3 germ layers
the TATA binding protein
binds to TATA box near position -30 of the promoter region
TATA box sequence
5’-TATAAAA
purpose of TATA box/protein
plays a major role in transcription initiation
how are TATA-binding proteins recruited
through proteins called TBP-associated factors (TAFs) that recognize other promoter sequences
structure of the TATA binding protien
Saddle-shaped protein that is able to create a sharp bend in the DNA upon binding
TBP sits on the DNA double helix like a saddle, with an extended beta sheet and loop stirrups in contact with the minor groove of the TATA box sequence
is the structure of the TATA binding protein conventional
No, it is unconventional - most DNA-binding proteins recognize DNA by inserting alpha helixes into the major groove
base pairs that are favoured in recognition sequence of TBP
A=T base pairs are favoured in the recognition sequence
More easily distorted to allow opening of the minor groove
Pol II promoters have variety of
transcription factors
1. TFIID (composed of TBP and TAFs)
2. FIIB
3. TFII proteins
purpose of EMSA and DNA footprinting
to determine the presence of a transcription factor
EMSA
electrophoretic mobility shift assay
how EMSA works
- Fragments of DNA of a known sequence are incubated with the protein of interest and then analyzed on a nondenaturing polyacrylamide gel
- Visualized by staining with a dye or covalently attacking a radioactive phosphate group at one end
- Free DNA fragments migrate more quickly through the gel than DNA bound by protein
- A shift in migration of DNA from fast to slow in the presence of protein indicates a direct binding interaction between the protein and DNA
EMSA results
an increase in protein concentration = DNA bound protein grows longer, as well as there is less unbound DNA
what gel is used for EMSA
nondenaturing, since the addition of SDS (gel used for SDS-PAGE) would denature the protein, and disrupt formation of the DNA-protein interaction
DNA footprinting goal
to map the exact nucleotide bases in contact with the bound protein once a direct DNA-binding interaction has been established
DNA footprinting steps
- DNA of interested is amplified and radiolabeled at one end. One of the two PCR primers must be radiolabeled on the 5’ end to provide a point of reference for visualization
- Cleavage occurs at sites that are not physically protected by bound proteins. Each piece of DNA is cleaved once, generating a set of fragments that represent all possible cleavage products (except DNA that is protected by the bound protein)
- Resulting fragments are separated by gel electrophoresis and detected by exposing the gel to film which detects radioactive emissions from the labeled DNA fragments. Any gaps (DNA protected by bound proteins) produces a “footprint” that indicates the boundaries of the protein-binding site relative to the radiolabeled end of the DNA sequence of interest
DNA footprinting results
footpring is identified by analyzing the sites of nuclease cleavage in the DNA before and after adding the protein
processing of pre-mRNA in prokaryotes
Can be directly transcribed, sometimes while still being synthesized
Directly translated by ribosomes
No compartmentalization (dividing into sections) of these processes
processing of pre-mRNA in eukaryotes
Transcription and pre-mRNA processing takes place in the nucleus
Mature mRNA has to exit the nucleus to the cytoplasm to be translated
structure of mature mRNA
5’ cap
3’ poly(A) tail
coding region - composed of the exons spliced together with no introns included
purpose of 5’capping
prevents degradation from the 5’ end
which polymerase helps with 5’capping and how
RNA Pol II synthesizes single-stranded ribonucleic acids that are vulnerable to degradation at either end by exoribonucleases
what is the 5’ cap made of
residue of 7-methyl-guanosine, 5’-5’-triphosphate linkage, and additional methyl groups
5’-5’-Triphosphate Linkage
7-Methyl-Guanosine is linked to the 5’-terminal residue of the mRNA through an unusual 5’,5’-triphosphate linkage
Generated by an enzyme called guanylyltransferase (capping enzyme) which forms the 5’ cap through condensation of a molecule of GTP
additional groups added to 5’ capping
methyl groups, added at the 2’ hydroxyl of the ribose sugars at the first and second nucleotides adjacent to the cap
steps of 3’Polyadenylation
- RNA polymerase will transcribe the poly(A) site at the terminal end of the transcript
- The mRNA site where cleavage occurs is marked by 2 sequence elements
- Highly conserved sequence 5’-AAUAAA located 10 to 30 nucleotides on the 5’ side (upstream) of the cleavage site
- A less-well defined sequence rich in G and U residues located 20-40 nucleotides downstream from the cleavage site - Polyadenylation factors bind the poly(A) signal upstream of the cleavage site
- Subsequent cleavage generates the free 3’-hydroxyl group that defines the end of the mRNA - A residues are immediately added to the frere 3’-OH of the reaction
- Bound by poly(A) binding proteins (PABPs) which physically protect the 3’ mRNA from exoribonucleases
major difference between mRNA processing in bacteria & eukaryotes
splicing of pre-mRNA
do prokaryotes have introns
no
alternative splicing
exons in the primary transcript from a single gene are spliced together in several combinations to produce different mRNAs = different polypeptides
what does alternative splicing give
different polypeptides = genetic diversity
oligo dT primers
using a poly(T) sequence as a primer in the reverse transcription reaction- 3’ poly(A) tail is not present on rRNA
characteristics of the genetic code
- Degenerate
- Several codons have the same meaning
- 64 unique ways to combine 4 different nucleotides in a triple codon sequence, but only 20 common amino acids
- Multiple codons encode for the same amino acid
- 61 coding amino acids, 3 specifies the termination of translation amino acids - Robust
- Degeneracy provides DNA with the ability to absorb single-base mutations with minimal consequences to the protein sequences it encodes - Universal
- All organisms use the same genetic code with a few minor modifications
- Once the code evolved, it resisted change
rules of the genetic code
- It is non-overlapping
- It does not contain pauses
- The genetic code is read in triplets
- It is read in a linear fashion
- The code is resistant to mutations
non-overlapping vs overlapping codons
non-overlapping: a single nucleotide substitution would change only 1 codon = 1 amino acid change
overlapping: a single nucleotide substitution would change 3 codons = protein would have 3 consecutive amino acid changes
the genetic code does not contain pauses experiment & result
Brenner and Crick used mutagens known as acridines to induce mutations in the B gene of the T4 bacteriophage - Acridines produce mutations through the insertion or deletion of a single base pair into DNA
result: Most mutations completely inactivated the B gene - Suggested that not 1 but many amino acids were changed by these mutations
frameshit mutation
insertion or deletion of 1 or more paired nucleotides, changing the reading frame of codons during protein synthesis
translation is linear rule experiment
looked at the synthesis of hemoglobin, using extracts from rabbit reticulocytes (immature red blood cells)
transition mutation
a purine is replaced by another purine or a pyrimidine is replaced by another pyrimidine
transition mutations positions
- third position
- rarely cause a change - second position
- determines whether it encodes a polar (purine) or hydrophobic (pyrimidine) amino acid
- tend to conserve the chemical nature - first position
- results in an amino acid change, but chemically similar to the original
silent mutation
Single-base substitutions
Non deleterious and non-harmful
Do NOT result in an amino acid replacement
missense mutation
Single-base substitution
Replaces one amino acid with another
Can be deleterious or non-deleterious
nonsense mutation
Far more harmful than silent mutations
Result in a termination codon
Abort protein synthesis
Results in an incomplete protein that is rarely functional
non-stop
loss of a stop codon
