Lecture #6 - The structure and function of mRNA

mRNA - transient messenger between gene and protein

Bacterial mRNA t½ ≈ 2 minutes
Eukaryotic mRNA t½ ≈ 6-24 hours
Probably a reflection of the environments

Only one strand of DNA is transcribed for one mRNA

5' 3‘ RNA-like strand (non-template strand)

3 5' Template strand
5' 3' mRNA

3'
5' 3‘ does NOT occur
3' 5‘
5’

5‘ 5’ 3‘ does occur
3' 5‘
5’

5'
5' 3' does occur
3' 5'

5'
Differences between prokaryotes and eukaryotes

Eukaryotes
1) mRNA is synthesized as a large precursor
2) After synthesis is complete mRNA transported to cytoplasm
3) mRNA translated in cytoplasm
4) Transcription -translation are not coupled
5) mRNA codes for only one polypeptide
6) Long-lived mRNA

Prokaryotes
1) Transcription and translation are virtually simultaneous
2) Short-lived mRNA
3) mRNA can code for many polypeptides
1) mRNA synthesized in 5'  3' direction
2) Since translation occurs in 5'  3' direction,
translation can begin before transcription is finished
3) Note different lengths of mRNA
The coupling of transcription, translation, and degradation of bacterial mRNA
1) Rate of transcription is ~42 nucleotides /sec
Note that these are the same rate
2) Rate of translation is ~15 codons /sec

3) For 1200 bp gene, mRNA synthesis takes 2.5 minutes,

protein appears after 3.0 minutes or 0.5 minutes later

4) Degradation begins even before synthesis of mRNA is completed

mRNA stability
1) Functional t½ is capacity to be translated; for bacterial mRNA t½ ≈2 minutes
2) Chemical stability is amount of RNA that can hybridize to DNA
Chemical t½ >> functional t½ because degradation starts with a cleavage
that is not detected by hybridization
Method of hybridization
Unlabeled DNA

Add to filter, immobilize labeled RNA

Mix with DNA excess

Count in scintillation counter

Note: method will not tell you about RNA signal

mRNA degradation

Endo-and exonucleases both are involved

1) Endonucleases start the process.

There are specific targets; without these targets the mRNA is more stable

2) Exonucleases degrade RNA in 3' 5' direction.

Clearly the 3' end is generated after cleavage by endonucleases

Certain sequences or structures can slow down the exonucleases

A typical bacterial mRNA

trp operon codes for mRNA which is translated into 5 peptides:

15 initiations per minute (transcription)
30 ribosomes per mRNA
Therefore 450 peptides/minute
75% of E. coli genes are in operons
 Initiation of translation in polycistronic mRNAs

AUG AUG

1) Intercistronic region can vary from -1 to +40 bases

For -1 ----NNNUGAUG----

Termination codon Initiation codon of gene 2

of gene 1

2) The same ribosome can translate two genes if the start and stop codons
are within 15 bases - half of mRNA protected by a ribosome
3) Different ribosomes can translate the different genes if the SD and AUG are accessible
4) The intercistronic region may contain sequences that impede exonucleases
5) Failure to translate the first cistron can result in failure to translate subsequent cistrons (polarity)
Isolation of mRNA

(to characterize mRNA structure or the protein product)

1) Bacterial mRNA
a) Label cells with 32 P-Uridine
b) Extract nucleic acids
c) mRNA and rRNA cannot be separated

2) Eukaryotic mRNA - Different

Labeling with Uridine does not work
a) Instead isolate polysomes (sucrose gradient)
b) Treat polysomes with EDTA to dissociate mRNA; mRNA is released,
large amounts of "contaminant RNA" is not released
c) The mRNA is actually a ribonucleoprotein particle
Polyadenylation of mRNA
1) Can be used to isolate mRNA

2) Poly (A) tail is added to 3' end by poly (A) polymerase in the nucleus
3) About 200 A's added to mRNA and is shortened in cytoplasm
4) Shortening the poly (A) tail does not affect translational efficiency or mRNA stability
5) 67% mRNA contains poly (A) tail
33% does not contain poly (A) 30% is histone mRNA

70% standard mRNA

 Translation of isolated mRNA

Slow
Inefficient

Proteins
hard to
purify

Difference between in vitro and in vivo translation indicates translational control

Frequently-used cell-free systems are
1) E. coli
2) Wheat germ
3) Rabbit reticulocyte
Major advantage of in vitro system is ability to isolate primary translation product.
Frequently the protein is processed, the initial methionine may be removed
Capping of Eukaryotic mRNAs
1) All Eukaryotic mRNA contain cap except in mitochondria and chloroplast

2) Capping reaction is Gppp + ppp A/G  Gppp A/G + PPi + Pi

3) The enzyme guanylyl transferase adds cap in the nucleus

4) Cap can be modified by methylation

I. OPERON: a unit of gene expression

-Contain more than one gene

-All genes of operons are transcribed as one unit
transcript is said to be POLYCISTRONIC
-75% of all genes in E. coli are in operons;
also, many viral genes are in operons, e.g., T4 rlIA & B
-Genes in operons tend to have a common function;
for example, lac genes metabolize lactose
-Yeast genes tend to be clustered, but they are MONOCISTRONIC
only bacterial genes have operons

II. Principles that emerged from the work of Jacob and Monod
1. Two types of genes
A. Structural: genes that code for an enzyme or structural protein

B. Regulatory: some genes code for proteins whose sole function

is to regulate gene expression

Operons may contain both types of genes

2. Many regulators bind DNA and regulate initiation of transcription;
some affect translation

Methods used to analyze DNA binding include footprinting,

methylation protection, and UV crosslinking

3. Regulatory proteins may exist in active and inactive conformations:

Interconversion may be by binding INDUCERS or CO-REPRESSORS;
by nucleotide exchange; or by covalent modification

III. Why the study of the lac operon was productive as a model
system for regulation

1. Combination of genetic and biochemical analyses were available

2. Enough was known to form testable hypotheses
3. lac operon is completely dispensable; therefore, exotic genetics
were not necessary
4. Mutants are easy to isolate, as are suppressors.
5. There are a number of useful analogs;
A. For assays
B. For induction
 Genes, mRNA and Proteins of the lac operon

Proteins
1) β-gal (lacZ): measured by cleavage of ONPG
2) Permease (lacY): measured by uptake of radioactive thiogalactosides, such as IPTG
3) Transacetylase (lacA): catalyzes the reaction
Acetyl-CoA + thiogalactoside  6-acetyl-thiogalactoside + CoA
This enzyme has no known function because thiogalactosides are not natural
Analogs
1) ONPG- orthronitro-β-D-galactoside
Cleaved 27X faster than lactose
Used for β-gal assay
Product, ONP, is intensely yellow
2) X-gal: 5-bromo-4-chloro-3-indolyl-β-D-galactoside
When in plates, colonies with β-gal are blue
colonies without β-gal are white
Transported by unknown system (not lacY)
X part insoluble
3) Melibiose: 6-O-α-D-galactopyranosyl-D-glucose
Transported by lacY
Not cleaved by lacZ
Therefore useful way to tell if cells are lacY+
Inducer
4) Raffinose: O-α-D-galactopyranosyl-(16)-O-α-D-glucopyranosyl;
-(12)-β-D-fructofuranoside
gal-glu-fru
Requires lacY
Not cleaved by lacZ
Not an inducer
Therefore grows only if lacY+ and lacI- or lacOc
5) IPTG: isopropyl-β-D-thiogalactoside
An excellent inducer (binds to repressor)
Not a substrate for β-gal
A gratuitous inducer
6) TONPG: orthronitrophenyl-β-D-thiogalactoside
Transported by lacY product
Toxic
Can select for Y- or lac-
(only in medium with succinate)
Evidence for operon model

1) When induced, all the genes expressed

2) A common regulator for all three (repressor)

3) A single site of regulation (operator)

4) A single promoter
Regulation

Expression of lacZYA controlled by regulator protein

LacI (lac repressor)

Gene located upstream of lac genes

Independent transcriptional unit

Trans-acting (diffusible)

Negative regulator

lac genes on all the time unless turned off by regulator

Tetramer that binds operator (Olac) sequence

Prevents RNP binding

Necessity for regulation

If substrate (β-galactosides) not available; why make metabolizing enzymes?

When substrate appears there is a need to make enzymes quickly

Induction

No β-galactosides  no enzyme (<5)

β-galactosides  enzymes made fast (2-3 min) (5000)

lac mRNA unstable

Induction

Executed by inducer
Substrate or product of the enzyme activity

Inducers can or cannot be metabolized by the enzyme they induce

Example:

IPTG (isopropylthiogalactoside)

Not metabolized by β-galactosidase

Very good inducer of lac genes

Gratuitous inducer  remain in the cell

Inducer acts on the repressor (lacI)

No inducer  lac genes off

Plus inducer  lac genes on

Repressor

Can bind operator and inducer

When bound to inducer  no operator binding

Uninducible  not expressed at all

Constitutive  No response to regulation

Promoter and operator mutations (cis-acting)

Repressor mutations (trans-acting)

Physiology of mutants

Repressor mutations

lacI- (i -)

Loss of function (deletion, non-sense, etc.)

No repression

Transcription constitutive

lac genes on all the time

Presence of inducer irrelevant

Exp.
i -z+y+/F' i+z-y+  regulation restored

Therefore  trans-acting
Mutations in the operator

Operator mutants are constitutive and are called oc

Unlike i - mutants, which are also constitutive, oc mutants are dominant to o+

oc mutants are dominant only on the chromosome that contains oc. For example:

i+ocz+y-/F' i+o+z-y+ Constitutive for β-gal, not for permease

i+o+z+y-/F' i+ocz-y+ Constitutive for permease, not for β-gal

oc mutations are said to be cis-dominant

Genetic evidence for the lac repressor; evidence that repressor is a protein
We know that repressor binds operator, which is between the promoter
and the lacZ gene

Inducer binds repressor and this complex fails to bind DNA

The following experiment demonstrated that the repressor existed in the cytoplasm:
the PaJaMo experiment (Pardee-Jacob-Monod)

During conjugation, cells are transiently diploid. The following crosses were performed:

1) Hfr i+z+strS X F-i -z-strR

β-gal was made for 90 minutes even without inducer. Then synthesis stopped.
The burst is called zygotic induction

2) A different cross was done in which the i+ and i - loci were switched

Hfr i -z+strS X F-i+z-strR

β-gal was not made!

The conclusions were:

1) No mixing of cytoplasm. If there was mixing, then the result would

be symmetric because the genotype of the mix would be i+/i –

2) The genetic trait, inducibility (i+) is present in the cytoplasm.

i+ makes a repressor; i - lacks the repressor

3) Synthesis of β-gal stops in cross 1 when sufficient repressor accumulates

Evidence the repressor is a protein

1) Some i - mutants were temperature sensitive

2) Some i - mutants had suppressible nonsense mutations

3) The repressor could be purified

 Filter-binding
Assay
The DNA will
be radioactive

The following experiments were done to prove that the repressor was purified
1) Protein binds wild-type operator DNA (o+)
2) Protein does not bind oc DNA
3) IPTG release repressor from o+ DNA
4) Repressor binding required double-stranded DNA
Curiously, lactose did not release from o+ DNA. The true inducer is allo-lactose,
a product of a side reaction between lactose and β-galactosidase.
This fact implies that induction also requires some β-galactosidase.
Other mutations

Plac  cis-acting
Could prevent RNP binding or enhance binding

lacI s  trans-acting
Repressor fails to bind inducer
Always in active form
Prevents transcription (uninducible)
iq overproduces repressor

Up mutation in promoter of lacI gene

Normally 10 molecules repressor per cell

iq have 100 molecules
iq' has 500 molecules

i -d bind IPTG (inducer), but not DNA

Trans-dominant constitutive  i-d/i+ are constitutive

because of subunit mixing

All i -d map to 1st 60 amino acids of lacI gene

Therefore DNA binding region maps there

 kB (M-1)
DNA Repressor Repressor + inducer
lac operator 2 x 1013 2 x 1010
other DNA 2 x 106 2 x 106
___________________________________________________
Specificity 107 104