6.4 Regulation at the Level of Translation - uni … · 6.4 Regulation at the Level of Translation...

6.4 Regulation at the Level of Translation

6.4.1 The components

6.4.2 Initiation

6.4.3 Elongation

6.4.4 Termination

6.4.5 Alternative translation events

6.4.6 Translational control of gene expression

6.4.7 Additional amino acids

6.4.8 Posttranslational regulation


6.4.2 Initiation

6.4.3 Elongation

6.4.4 Termination





The Components

1. The tRNAs 2. The aminoacyl-tRNA synthetases3. The 70S ribosomes 4. The mRNAs

The tRNAs

1. They translate individual codons of the mRNA into the corresponding amino acid

2. They are recognized and aminoacylated specifically by their cognate aminoacyl-tRNA synthetases

3. They show a conserved cloverleaf-shaped secondary structure that folds into an L-shaped tertiary structure

4. Average length: 76 nucleotides

The Aminoacylation Reaction

First step: Activation at the active site with ATPSecond step: Transfer to the 3‘ end of the tRNA

J Ling (2009) Annu Rev Microbiol 63: 61

The Two Classes of Aminoacyl-tRNA Synthetases

Class I Class II

CF Francklyn (2008) Biochemistry 47: 11695

The 70S Ribosome

30S ribosomal subunit:- 16S RNA- 21 proteins

50S ribosomal subunit:- 5S and 23S RNA- 34 proteins

Three sites called A, P and E

Peptidyltransferase center: Start of an 100 Å long tunnel, 15 Å diameter

The mRNA

Two types of mRNAs:CanonicalLeaderless

Canonical mRNA: Contains an 5' untranslated region (5' UTR) with

Shine-Dalgarno (SD) sequenceStart codonS1 binding site (pyrimidine-rich)Sometimes regulatory sequences

Leaderless mRNA:Starts with the start codon or a few nucleotidesupstreamThere is no SD sequence

I Moll (2002) Mol. Microbiol. 43: 239

Downstream Box (DB):

Occurs downstream from the initiation codonCan potentially base-pair with a region within the16S rRNANo genetic and biochemical evidence for thisinteraction


6.4.2 Initiation

6.4.3 Elongation

6.4.4 Termination





The Prokaryotic Translation Initiation Region (TIR)


6.4.2 Initiation

6.4.3 Elongation

6.4.4 Termination





Two mechanisms compete with

unimpaired elongation:

1. Drop-off

2. SsrA-tagging

Minigenes in Phage Lambda DNA

What happens when translating ribosomes arrive at the 3' end of a mRNA molecule devoid of its stop

codon ?

Will they dissociate spontaneously ? Will they stay stalled forever ? Is there a mechanism to release them from the mRNA ?

By which mechanisms mRNA molecules without a stop codon can

arise ?

Premature transcription termination

Endoribonucleolytic cleavage

The SsrA-Tagging Mechanism

Physiological roles of the tmRNA-

dependent peptide-tagging system:

1. Clearance and recycling of stalled ribosomes

2. Degradation of abortive proteins by cellular ATP-dependent proteases

Trans-Translation Occurs in a Number of Different Circumstances

(B) no stop codon(C) rare codons(D) at a stop codon(E) ribosomes blocked

by peptidyl transferase

(F) inhibitors such a spectinomycin

Model for Trans-Translation Acting on the lacI mRNA

T Abo (2000) EMBO 19: 3762

Translational Coupling

Definition:Translation of the downstream genes needs translation of the upstream gene

Direct coupling:SD or/and initiation codon of the downstream gene is part of a secondary structure of the mRNA which is destroyed by translating ribosomes

Mechanisms of Translational Coupling

Indirect coupling:If the upstream gene cannot become completely translated, the downstream gene will be inactivated, e.g., by an endonuclease

Biological Function of Translational

Coupling

1. Ribosomes will not dissociate at the first stop codon

2. Ensures synthesis of equimolar amounts of the two proteins


6.4.2 Initiation

6.4.3 Elongation

6.4.4 Termination





Components of Translation

Termination

1. Three stop codons: UAG, UGA, UAA

2. Four release factors: RF1, RF2, RF3, RRF (RF4)

Function of the Four Release Factors

RF1: decodes UAG and UAA

RF2: decodes UGA and UAA

RF3: catalyzes dissociation of RF1 and RF2 from the ribosome

RF4: catalyzes dissociation of the ribosomal subunits

Termination of Translation and Ribosome Recycling in E. coli

S Petry (2008) Curr. Op. Struct. Biol. 18: 70


6.4.2 Initiation

6.4.3 Elongation

6.4.4 Termination





Read-through:1. A tRNA decodes the stop codon2. Translation continues until the next stop codon

Frameshifting:Ribosome switches to one of the two alternative frames

Bypassing:Extensive peptidyl-tRNA slippage and re-pairing with the mRNA at a non-overlapping codon

Alternative Translation Events

1. Autoregulation: To limit the amount of a protein, e.g., RF2

2. To yield two different proteins, e.g, dnaXgene codes for the two proteins τ and γ

Biological Functions of Frameshifting

Pausing site

Slippery (shifty) sequence

Components of Programmed

Frameshifting

1. Termination codon

2. Stem-loop structure

3. Pseudoknot

4. Hungry codon

Pausing site can be

Autoregulation of RF2 Synthesis by +1 Frameshifting

O Namy (2004) Mol. Cell 13: 157

Shifty sequence

Frameshifting in the E. coli dnaXGene

In-frame product: γ-subunit; 47 kDaTrans-frame product: τ-subunit; 71 kDaSlippery sequence: A AAA AAGSlippage from AAA GAG to AAG AGU

Definition:

Joins the information found within two disparate open reading frames into a single polypeptide chain

Translational Bypassing

Examples of Translational Bypassing

Take-off codon: boxedLanding-site codon: underlined

A: +5 bypassing

B: +6 bypassing

C: +2 bypassing

D: T4 gene 60 bypass

E: starvation bypass „hungry“codon

A Model for T4 Gene 60 Bypassing

Peptidyl-tRNAGly

detaches from the take-off site GGASlides through the decoding centerRe-pairs at the landing site GGAresumes translation~50% of the ribosomes traverse the coding gap successfully

Topoisomerase subunit

AJ Herr (2000) EMBO J. 19: 2671

1. The matching GGA codons2. An optimal spacing of 47 nucleotides

between the two GGA codons3. A nascent peptide signal4. A stem-loop structure just downstream of

the take-off site5. A UAG stop codon6. An SD-like interaction

Signals involved in bypassing:


6.4.2 Initiation

6.4.3 Elongation

6.4.4 Termination





Translational control by

activation:

1. Activator RNA2. Activator protein3. cis-acting RNA element

DsrA Activates Translation of rpoS

mRNA

rpoH mRNA

Structure

Acts as a

Thermometer

PJ Schlax (2003) Mol. Microbiol. 48:

1157

Translational control by repression:

1. Antisense RNA2. Repressor protein

Antisense RNA Mediated Repression

S Chen (2004) J. Bacteriol. 186: 6689

Example:

Inhibition of translation of hns mRNA

S Brantl (2002) BBA 1575: 15

Protein-Mediated Translational

Repression Mechanisms

1. Direct competition model

2. Entrapment model

Entrapment Model of Translational Repression

PJ Schlax (2001) J. Biol. Chem. 276: 38494


6.4.2 Initiation

6.4.3 Elongation

6.4.4 Termination





Summary of Genetic Code Changes

A Ambrogelly(2007) Nature

Chem Biol 3: 29

Is a stop codon always a stop codon ?

Classical stop codons: UAA, UAG, UGA

1. Mitochondrial genomes UGA = tryptophan codon tRNATrp recognizes both UGG and UGA

2. Mycoplasma capricolumUGA = tryptophan codon

3. Many eubacteria and archaeaUGA = selenocystein (Sel) UGA = pyrrolysine (Pyl)

There are two additional amino

acids found with proteins:

Selenocysteine (Sel) Pyrrolysine (Pyl)

Properties of the Amino Acids

Seleno-cysteine and Cysteine

L Johansson (2005) BBA, in press

1. Selenium is a trace element incorporated into proteins in the form of selenocysteine (Sec) in all three kingdoms

2. Found mainly in the active site of oxidoreductases; negatively charged at physiological pH and highly reactive

3. Incorporated into proteins during translation elongation

4. Encoded by a combination of internal UGA stop codon and a specific mRNA hairpin structure = SECIS (selenocysteine insertion element)

Selenocysteine

The Four sel Genes of E. coli

selA: Selenocysteine synthetase; converts the seryl moiety into the selenocysteyl derivative

selB: specific elongation factor (EF-Tu)selC: tRNASec; charged with L-serine and serves

as an adapter at which the seryl moiety is converted into the selenocysteyl derivative

selD: Selenophosphate synthetase; synthesizes monoselenophosphate from selenide and ATP

The specialized SelB elongation factor binds three different ligands:

1. GTP2. Sec-tRNASec

3. The SECIS element (in bacteria located immediately 3' of the UGA codon)

In E. coli, three formate

dehydrogenases contain selenium:

1. fdoG: expressed constitutively2. fdhf: expressed under anaerobic conditions3. fdnG: expressed under anaerobic conditions

Two SECIS Element

fdhF: TGA CACGGCCCUTCGGUUGCAGGU...

M Thanbichler (2002) EMBO J. 21: 6925

Proposed Model of the Reactions Involved in the Regulation of selAB

Expression

Pyrrolysine Methanosarcina barkeriUAGPresent in the active site of methogenicmethylaminemethyltransferase (catabolizes methylamineto methane)Specialized tRNACUA and lysyl-tRNA synthetasePyrrolysine: lysine in amide-linkage to (4R,5R)-4-substituted-pyrrolysine-5-carboxylate

Putative Scheme for the Cotranslational Insertion of

Pyrrolysine at Amber Codons

M. Ibba (2002) Current Biology 12: R464

Distribution of Sel and Pyl

Deltaproteobacterium δ1:57 selenoprotein genes several Pyl-containing proteins


6.4.2 Initiation

6.4.3 Elongation

6.4.4 Termination





6.4.9 Collaborative behaviour in bacteria

1941: G. Beadle and E. Tatum

„ONE GENE - ONE ENZYME“ Hypothesis

George Beadle1903 - 1989

Edward Tatum1909 - 1975

Nobel Price 1958

Medicine

1. Overlapping genes

2. Ribosomal frameshifting

3. Bypassing

4. Gene within a gene

5. Polyprotein processing

Mechanisms Involved in Creating

Multiple Protein Products from a

Single mRNA

Overlapping Genes

There are two classes:

In-frame overlapping genes: permit the synthesis of more than one form of the protein from the same gene

Out-of-frame overlapping genes

ZI Johnson (2004) Genome Res. 14: 2268

In-Frame Overlapping Genes

Overlap may beLimited Complete

Reasons for overlapping genes:Economy strategy (genome compactness) Transcriptional or functional coupling

First in-frame gene pair: genes A and A* of phage ΦX174M. genitalium: 162 overlapping genes M. pneumoniae: 203 overlapping genes

Polyproteins

Definition:A precursor protein is proteolytically cleaved in two or more proteins

E. coli Penicillin G Acylase

Enzyme catalyses the conversion of penicillin G to phenylacetic acid and 6-aminopenicillanic acid (precursor for semisynthetic penicillins)

1 3 2

L Thöny-Meyer (1992) FEBS Lett. 307: 62

B. japonicum cytochrome bc1

Characteristics of a signal sequence

B. subtilis spore coat proteins

S Cutting (1991) J. Bacteriol. 173: 2915

B. polymyxa Amylases

N Uozumi (1989) J. Bacteriol. 171: 375

Three different β-amylases: 70, 65 and 42 kDa

Porphyromonas gingivalis genes rgpA and kgp

Gram-negative, anaerobic bacteriumAssociated with human periodontitis

PD Veith et al. (2004) Infect. Immun. 72: 3655

What selective pressure forced evolution and maintenance of

polyproteins ?

Strict stoichiometry of the processed proteins

The different domains may fold interdependently (no chaperones needed)

Increase the local concentration of the different components (less dependent on diffusion)

There are two classes of circular proteins:

1. Those involved in host defense

2. Pilins

Circular Proteins

Three-Dimensional Structures of Naturally Occurring Circular Defense Proteins

Bacteriocin AS-48 (E. faecalis)

Microcin J25 (E. coli)

MCoTI-II (melon seed)

RTD-1 (leukocytes Rhesus)

kalata (Rubiaceae +

Violaceae)

SFTI-1 (seeds from sunflower)

Circular Defense Proteins

1. Range in size from 14 to 70 amino acids2. Involved in host defense3. Bacteriocin AS-48: Highly basic 70-amino acid

protein; forms pores in the cytoplasmic membrane of sensitive cells; thermal denaturation temperature = 93°C

4. They all appear to be derived from longer precursor proteins

5. Cycling enzymes or mechanisms of cycling unknown

Gene Arrangements and Biosynthesis of Microcin J25

Moonlighting Proteins

Definition:

Proteins with more than one function

Examples:

PepA: - endopeptidase- DNA-binding protein

BirA: - biotin ligase- DNA-binding protein


6.4.2 Initiation

6.4.3 Elongation

6.4.4 Termination





6.4.9 Collaborative behaviour in bacteria

Collaborative Behaviour in Bacteria

Definition:Groups of genetically identical organisms synchronize their patterns of gene expression to achieve specific goals that are unattainable for single cells acting on their own

Depends on cell-cell communication with secreted signaling molecules

Cannibalism and Fratricide

Definition:Some cells of a population produce a toxin which kills other cells of the population which do not produce the toxin (fratricide) and feed on them (cannibalism).

Two examples:

Sporulating Bacillus subtilis cells = cannibalism

Competent Streptococcus pneumoniae cells = fratricide

Cannibalism

The Sporulation

Pathway

JP Clavergus (2007) Nature Rev. Microbiol.

5: 219

Sporulation is Induced by

Nutritional Stress

kinase Akinase Bkinase C ⇒ Spo0F ⇒ Spo0B ⇒ Spo0A~Pkinase D | |kinase E RapA PhrA Spo0E

RapB PhrCRapE PhrE

phosphorelay

Molecular Mechanism of Cannibalism

The skf (sporulation killing factor) and sdp(sporulating delay protein) operons are turned on by Spo0A~P

skfA-H: eight genes

The skf Operon

skfA-H: eight genesskfA: 55 amino-acid bacteriocin-like peptide = killing factor ?skfEF: ABC transporter = export of SkfAskfD: integral membrane protein = involved in maturation of SkfA ?skfB: involved in post-translationally modification of SkfA ?

The sdp Operon

sdpABCsdpC: codes for the toxin; derived from the 63 C-terminal amino acids of SdpC

Both SkfA and SdpC belong to the group of antimicrobial peptides

Spo0A

DNA-binding protein acting both as a repressor and as an activator

Present in an inactive (Spo0A) and in an active form (Spo0A~P)

60-70% of the cells produce a high amount of Spo0A~P = Spo0A-ON30-40% of the cells produce a low amountof Spo0A~P = Spo0A-OFF

Cannibalism

Fratricide

Competence

JP Clavergus(2007) Nature

Rev. Microbiol. 5: 219

Induction of Competence

Competence develops in response to environmental signals such as- a change in external pH- antibiotic-induced stress:

Aminoglycoside antibiotics: Block ribosomefunction

Fluoroquinolone: Inhibits topoisomeraseMitomycin C: DNA damaging agent

Induction of competence = general stress response

Transformation of the Gram-

positive bacterium Streptococcus

pneumoniae

Induction of competence involves cell-cell communication mediated by the

competence stimulating peptide = CSP

Activation of Early com Genes

1. The secretion apparatus ComA/ComB isinvolved in secretion of the 17-residues CSP

2. If the cell concentration is high enough, CSPbinds to the ComD histidine kinase

3. Autophosphorylated ComD~P transfers thephosphate group to the response regulatorComE

4. ComE~P activates expression of ~20 early com genes among them comX

Activation of Late com Genes

1. comX codes for an alternative sigma factor2. ComX controls expression of ~60 late com

genes3. Four of these genes code for killing factors

- CbpD: murein hydrolyse- LytA: murein hydrolase - LytC: hydrolytic enzyme (not ComX-dependent)

- CibA, CibB: two-peptide bacteriocin

Fratricide

Tentative Model for Fratricide

Fratricide needs the three lytic enzymes and the two peptide bacteriocins

Fratricide is involved in cell lysis in trans = allolysis

Fratricide can still occur even if lytA and lytCare both inactivated provided they are intact in the targeted cells

How do cells prevent suicide ?

Two immunity proteins have been identifiedBoth are integral membrane proteinsCibC: 65 residues; two transmembrane segments; mechanism unknownComM: 206 residues

Who is killed ?

Competent cells kill non-competent cells

How could mixed pneumococcal populations arise ?

unknown

What is the biological function of

fratricide ?

Release of virulence factors and inflammatory

mediators

Access to DNA

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