Molecular Exam 4

3’ and 5’ UTR

The untranslated sequence downstream/upstream from the coding region of an mRNA

What is the difference structurally between prokaryotic, eukaryotic, and histone mRNAs?

prokaryotes - no cap or tail, stem-loop structure and/or poly uracils at the end

eukaryotes- 5 cap and polyA tail with polyA binding proteins, could be multiple stem loops of different sizes

histone - cap but no polyA tail, instead stem loop structure. Histone mRNA is made during S phase

ribonucleases

There are endo and exo. They lead to mRNA instability

Half life

Determines how long a mRNA lasts before half of them are biologically not functional

Steady state (molecular concentration)

The concentration of population of molecules when the rate of synthesis and degradation are constant

mRNP proteins

assist mRNA in the cytoplasm

RNA-binding proteins (RBP)

Exist in large numbers. Most remain uncharacterized

RNA regulons

mRNAs associated with regulatory proteins, which influence translation, localization, and stability of mRNA

Regulon classes are determined by mRNA sharing the same RNA Binding Proteins (RBP)

Polyribosome (polysome)

An mRNA that is simultaneously being translated by multiple ribosomes

How is prokaryotic mRNA degraded?

Initiated by removal of pyrophosphate from 5’ terminus

mRNAs are degraded in a two-step cycle involving endonucleolytic cleavages conducted by RNase E and followed by 3’-5’ digestion of resulting fragments

poly (A) nucleases

enzymes that degrade the poly-a tail via deadenylation

What are the two routes in which eukaryotic mRNA is degraded?

Lsm 1-7 stabilizes the 3’end of mRNA

Decapping protein (Dcp) removes the 5methylguanosine cap (endo)

Xrn1 digests nucleotides (exo) until it hits Lsm

Exosome catalyzes 3’-5’ mRNA digestion and is a evolutionarily conserved complex

Processing bodies (PBs)

Cytoplasmic particles where mRNA degradation may occur

Nonsense-mediated decay (NMD)

Targets mRNAs with premature stop codons using UPF and SMG proteins

What are two ways in which a premature stop codon is recognized in mRNA

1. Unusual length of 3’ UTR region

2. presence of downstream exon junction complexes in mammals

Nonstop decay (NSD)

Targets mRNA lacking an in-frame termination codon and requires a conserved set of SKI proteins

No-go decay (NGD)

Targets mRNAs with stalled ribosomes in their coding regions (studied only in yeast)

What all are included in Cytoplasmic Surveillance Systems?

1. Nonsense Mediated decay

2. nonstop decay

3. no-go decay

What are the three mechanisms for the localization of mRNA currently documented?

mRNA is actively transported, via microtubules, to site of translation (most common mechanism)

mRNA is uniformly distributed, but gets degraded at most sites, except site of translation (rare)

mRNA gets captured, and moved to the site of translation (rare)

ribozyme

An RNA that has catalytic activity

RNA editing

A change of sequence at the level of RNA following transcription

What group of introns undertake self-splicing?

Group I introns

What factors are needed for auto splicing in vitro?

Two metal ions (Mg++ and Mn++) - They provide energy to be able to have the RNA in a certain conformation for the auto-splicing to occur

A guanosine nucleotide cofactor - Assists chemically with the transecterifications

How does the guanosine cofactor assist with self splicing and where are the guanosines

1. 3’-OH of guanosine attacks 5’ end of intron in the first transesterification (guanosine on linear intron)

2. The 3’-OH end generated at the end of the first exon attacks the junction between the intron and second exon in the second transesterification (exons connect)

3. The intron is released that cirularizes when its 3’-OH terminus attacks a bond at one of two internal positions in third transesterification (guanosine on 15bp segment that was broken off)

How does RNA editing work in Apolipoprotein-B gene?

Apolipoprotein-B and glutamate receptor mRNAs have site-specific deaminations catalyzed by cytidine and adenosine deaminases

C > U

or

A > I (which reads as G)

This allows for different proteins to exist in the liver versus intestine. It allows variety without having to write a bunch of mRNAs

How does RNA editing work in the trypanosome mitochondria?

There are various insertions and deletions of uridine

This happens with the pairing of a guide mRNA

Used for environmental stressors

Ribosome

Large ribonucleoprotein particles that contain more RNA than proteins and dissociate into large and small subunits

What are the different sites for tRNA in the ribosome?

An aminoacyl-tRNA enters the A site

Peptidyl-tRNA is bound to the P sites

Deacylated tRNA exits via the E site

Translocation

An amino acid added to a polypeptide chain by transferring the polypeptide from the peptidyl-tRNA in the P site to aminoacyl-tRNA in the A site

Initiation

The stages of translation up to synthesis of the first peptide bond of the polypeptide (slow)

elongation

the stage of translation in which the polypeptide chain is extended by the addition of individual subunits (faster process)

Termination

A separate reaction that ends translation by stopping the addition of subunits and causing disassembly of the synthetic apparatus

ribosome-binding site

A sequence on bacterial mRNA (that includes an initiation codon) that is bound by a 30S subunit in the initiation phase of polypeptide translation

Shine-Dalgarno sequence

the polypurine sequence AGGAGG centered about 10 nt before the AUG start codon on bacterial mRNA

It is complementary to the sequence at the 3’ end of 16S rRNA

What experiment was done to confirm the Shine-Dalgarno sequence?

Have the initiation sequence bond with the ribosome and then have nucleases get rid of the unprotected mRNA and leave the initial sequence

What is the difference between methionine tRNAs for proteins and for initiation in bacterial and mitochondrial proteins?

Methonine is the typical amino acid, but N-formyl-methionine is the modified amino acid for initiation

How does initiation occur in eukaryotic mRNA?

40S subunits bind to the 5’ end of the mRNA and scan the mRNA until they find the initiation site

The initiation site consists of a 10-nucleotide sequence that includes an AUG codon (Kozak sequence)

Then 60S ribosomal subunits join the complex at the initiation site

peptidyl transferase

ribozyme in the ribosome that catalyzes peptide bond formation during protein synthesis

What chemical reaction occurs to transfer one amino acid to the polypeptide chain?

The nitrogen in the amino acid bonds to the carbon in the peptide chain leave a hydroxyl group (transferring an H from A site to P site tRNA)

What recognizes the stop codons in bacteria?

Protein release factors

RF1

The bacterial release factor that recognizes UAA and UAG as signals to terminate polypeptide translation

RF2

The bacterial release factor recognizes UAA and UGA as signals to terminate polypeptide translation

Which subunit has the peptidyl transferase activity in prokaryotes and eukaryotes?

23S rRNA and 28S rRNA

What are the stop codons?

UAA (ochre) UAG (amber) UGA (opal)

Coupled transcription/translation

Transcription and translation occur simultaneously (bacteria)

cistron

An opening reading frame coding for a specific polypeptide

Monocistronic mRNA

a mRNA that encodes one polypeptide

Polycistronic mRNA

A bacterial mRNA containing several non-overlapping coding regions that represent different cistrons

Intercistronic region

The distance between the termination codon of one gene and the initiation codon of the next gene

third-base degeneracy aka wobble

The nucleotide present in the third position of some codons can be variable

Wobble allows G-U pairs to form

tRNA base modification

Involves direct alteration of the primary bases in tRNA. There are certain spots where wobble can happen like site 34 or 37

What are some pros and cons of tRNA modifications

Pros: Tune the precise codon reading, stabilize codon-anticodon interactions, allow tRNA to rapidly react to environmental changes (stress)

Cons: Many disorders come from it like Type II diabetes, familial disautonomia, X-linked intellectual disability

Inosine

Inosine is a modified base that can pair with uracil, cytosine, and adenine

2-thiouridine

A modified base that makes the uracil base only pair with adenine and stop wobble pairing with guanine

Suppressor tRNA

a tRNA that has a mutation in the anticodon that changes the codons that it recognizes

Nonsense suppression

Nonsense mutations can be suppressed by a tRNA with a mutant anticodon

Missense suppression

Occurs when the tRNA recognizes a different codon from usual so that one amino acid is substituted for another

What are cons to suppressor tRNAs?

Missense suppressor tRNAs compete with wild type tRNAs

Efficient nonsense suppression may cause readthrough past normal termination codons

How does 16S rRNA influence accuracy of translation?

There are interactions between N on sites 1492-1493 on rRNA and the N1 position of the nucleoside ring. If those interactions cannot occur, then the incorrect tRNA diffuses out

operon

A unit of bacterial gene expression and regulation, including structural genes and control elements in DNA recognized by regulator gene products

regulator gene

A gene that encodes a product (typically protein) that controls the expression of other genes

structural gene

A gene that encodes any RNA or protein product other than a regulator

Operator

The site at which regulator proteins interact with the operon.

negative regulation (operons)

A repressor protein binds to an operator to prevent a gene from being expressed: default setting is gene is “on”

Positive regulation (operons)

A transcription factor is required to bind at the promoter in order to enable RNA polymerase to initiate transcription

Inducible regulation

expression of a gene is regulated by the presence/absence of an inducer that is a substrate of the gene’s product

catabolic enzymes

specialized proteins that break down complex molecules into simpler ones, releasing energy for cellular processes

Repressible regulation

Expression of a gene is regulated by a corepressor that is the product of its enzyme pathway

Anabolic enzymes

biological catalysts that facilitate the synthesis of complex molecules from simpler ones

What are the four possibilities for in vivo gene regulation?

negative inducible (lac) - repressor protein, gene usually off

negative repressible (trp) - repressor protein, gene usually on

positive inducible (lac) - activator protein, gene usually off

positive repressible - activator protein, gene usually on

What happens if one of the structural genes in the lac operon is mutated?

If one of the genes mutates, then the whole thing falls apart

What are the lac operon genes and what does each one do?

lacI - gene for repressor protein. Repressor protein binds to operator

lacZ - gene for beta-galactosidase. The enzyme that separates the glucose monomer from the galactose

lacY - gene for permease. Goes outside the cell to let in lactose

lacA - gene for transacetylase. Has to modify the lactose before the enzyme can break the bond between sugars

How does the repressor get removed from the lac operon?

Lactose causes allosteric interactions with the repressor causing it to change shape and jump off the operator

How does lactose get in to remove the repressor if the gene to let in lactose is being repressed?

It is not a 100% repression; there is still a basal level of transcription happening that will allow permease to be made and let in lactose

The lac mRNA is extremely unstable, so what does induction look like in regard to amount of lac mRNA and level of beta-galactosidase?

lac mRNA - Makes almost a bell curve- with mRNA taking a second to catch up with inducer being added and removed

beta-galactosidase - Takes a second to reach induced level then stays that way for a bit since proteins have a longer half-life than mRNA

What happens if there is a mutation in the operator of the lac operon?

Causes the constitutive expression of all three lac structural genes. It is a cis-acting mutation

What if there is a mutation in the promoter of the lac operon?

This prevents expression of the lac operon, so they are uninducible. This is also a cis-acting mutation

What is a merodiploid e coli cell?

An e coli cell that has its host genome, along with a plasmid with mutant genes, so it could be diploid for some genes, but not all genes

interallelic complementation

The change in the properties of a heteromultimeric protein brought about by the interaction of subunits coded by two different mutant alleles

How is a superrepressor formed in the lac operon?

If a mutation causes the inducer-binding site to not work on the repressor

What is lacI -d

It is the dominant negative mutation of lacI. This is the situation where if one subunit of the tetramer is mutant, then the whole tetramer will not work

negative complementation

This occurs when interallelic complementation allows a mutant subunit to suppress the activity of a wild-type subunit in a multimeric protein

How does the repressor interact with the operator?

The operator is a sequence that is imperfectly palindromic, so each of the two subunits will grab either end of the palindrome and bend the DNA so transcription cannot occur.

Once the inducers bind to the repressor, it reduces the affinity of the repressor to the DNA

catabolite repression

The ability of glucose (the catabolite) to prevent the expression of a number of genes

Catabolite repressor protein (CRP)

An activator protein (a positive regulator) that binds to a target sequence at a promoter

What if there is a mutation in the crp gene?

Then there would be no activator, so even with low glucose, the activator would not be able to place the RNAP