Microbial Physiology

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Microbial Physiology

• Lecture 7

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Nucleic Acid Structure
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DNA
Nucleotide monomers
can be linked together via a phosphodiester
linkage
formed between the 3' -OH of a nucleotide
and the phosphate of the next nucleotide.
Two ends of the resulting poly- or
oligonucleotide are defined
The 5' end lacks a nucleotide at the 5' position,
and the 3' end lacks a nucleotide at the 3' end
position.
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DNA strands

• The antiparallel strands of DNA are not
  identical, but are complementary.
• This means that they are positioned to align
  complementary base pairs C with G, and A with T.
  
• So you can predict the sequence of one strand
  given the sequence of its complement.
• Useful for information storage and transfer!
• Note sequence conventionally is given from the 5'
  to 3' end

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RNA
Nucleotide monomers
can be linked together via a phosphodiester
linkage
formed between the 3' -OH of a nucleotide
and the phosphate of the next nucleotide.
Two ends of the resulting poly- or
oligonucleotide are defined
The 5' end lacks a nucleotide at the 5' position,
and the 3' end lacks a nucleotide at the 3' end
position.
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RNA has a Rich and Varied Structure
Watson-Crick base pairs (helical seg-ments
usually A-form). Helix is secondary
structure. Note A-U pairs in RNA
DNA can also form structures like this.
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Central Dogma of Biology
Replication
Translation
Transcription
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DNA Replication
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Origins of DNA Replication

• DNA replication begins from specific nucleotide
  sequences called origins of replication
• recognized by origin recognition proteins that
  open the helix and recruit the replication
  machinery
• DNA synthesis proceeds in both directions outward
  from the origin
• replicated double helices being produced
  ultimately join each other
• when complete, there are two identical daughter
  molecules

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DNA Synthesis
DNA chain growth
Replication fork growth
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5
3
5
DNA chain growth
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DNA replication is semiconservative
Replication is semiconservative, with each DNA
strand serving as template for synthesis of the
complementary strand
Fork movement
Replication fork
This is true for all eukaryotes, prokaryotes,
viruses and bacteriophage
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Leading and Lagging Strands

• Limitation is imposed by synthesis of DNA in a 5
  to 3 direction only
• The two DNA strands are used differently at
  replication fork
• leading strand is used for continuous DNA
  synthesis
• lagging strand is used in discontinuous synthesis
• forms Okazaki fragments
• fragments joined by DNA ligase

leading
lagging
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Lagging strand synthesis
Must supply a primer (i.e. 3-OH) to start DNA
synthesis This is the function of primase which
makes RNA primers
Must seal the DNA fragments made on the lagging
strand template This is the function of DNA ligase
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Replication Machine
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Transcription
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Prokaryote
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Initiation (Promoter) and Termination Signals for
Transcription
Prokaryote
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Prokaryote
Promoter
Cistron1
Cistron2
CistronN
Terminator
Transcription
RNA Polymerase
mRNA 5
3
1
2
N
N
N
C
N
C
C
1
2
3
Polypeptides
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Eukaryote
RNA polymerase II initiation
1. Binding of TFIID to the TATA box
2. Binding of TFIIA and TFIIB to TFIID on the DNA
3. Binding of RNA polymerase II complexed with
TFIIF
4. Binding of remaining general transcription
factors to assemble complex competent for
transcription
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Review of Eukaryotic mRNA Production
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Protein Synthesis
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The Codon
mRNA sequence is decoded in sets of three
nucleotides. Since there are 64 possible
tri-nucleotide combinations and only 20 amino
acids, there must be some redundancy (a.k.a
degenerate code).
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More amino acids

• 21st amino acid selenocysteine. Sometimes
  inserted at UGA stop codons.
• 22nd amino acid pyrrolysine. sometimes inserted
  at UAG stop codons.

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Reading Frames
A reading frame is the uninterrupted sequence of
codons from start to stop that encodes a sequence
of amino acids.
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Start Methionine
AUG is start signal for most proteins,
specifying an N-terminal methionine
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tRNA Needed

• tRNA or transfer RNA is needed for translating
  the codon to amino acid sequence.
• The tRNA is linked to an amino acid by an
  amino-acyl tRNA synthetase.
• There are two tRNAs for Methionine, of which only
  the initiation-specific tRNA can be used to start
  translation.

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tRNA
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Amino acid attachment to tRNA
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codon - anticodon
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Wobble

• Due to non-standard interactions, some tRNAs
  can base-pair with just two complementary bases.
• The third position can be mismatched (wobble).
• This allows the 61 codons to be matched by as few
  as 31 different tRNAs.
• In part, this can be due to non-standard bases,
  such as inosine, which is de-aminated adenine and
  can base-pair with A, C, and U. In addition, G
  can basepair with U, although its normal partner
  is C.

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• Wobble hypothesis
• Proposed by Francis Crick in 1966.
• Occurs at 3 end of codon/5 end of anti-codon.
• Result of arrangement of H-bonds of base pairs at
  the 3rd pos.
• Degeneracy of the code is such that wobble always
  results in translation of the same amino acid.
• Complete set of codons can be read by fewer than
  61 tRNAs.

Fig. 6.9
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All possible base pairings at the wobble position
No purine-purine or pyrimidine-pyrimidine base
pairs are allowed as ribose distances would be
incorrect (Neat!).
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Wobble pairing non Waston-crick base paring
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tRNA is not enough

• The tRNA can only base-pair with a complementary
  codon and bring an amino acid along. However, it
  cannot create a polypeptide alone.
• Ribosomes are required as the amino-acid-linking
  machinery.
• Ribosomes are multi-subunit structures consisting
  of both multiple rRNAs and proteins.
• Functionally can be considered two subunits,
  large and small (50S/30S in prokaryotes, 60S/40S
  in eukaryotes).

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Ribosome
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Ribosomal RNAs

• While their exact sequences differ, analogous
  rRNAs from different species all fold into
  similar structures containing many stem-loops and
  binding sites for other RNAs and proteins.
• It is thought that the large rRNA binds incoming
  tRNAs and catalyzes peptide bond formation
  (peptidyl transferase activity).

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Prokaryotic Initiation
Initiation factors bind 30S ribosomal subunit.
In conjunction with fMet-tRNAiMet, binds to site
near AUG.
Shine-Dalgarno sequence conserved 6 nucleotides
just upstream of start AUG. Basepairs with 3 end
of 16S rRNA.
Used as a basic translational control mechanism
in prokaryotes.
Large subunit binds in reaction requiring
hydrolysis of GTP bound to IF2
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Shine-Delgarno element
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Formation of initiation complex
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Eukaryotic Initiation 1
eIF6 and eIF3 serve to keep the 60S and
40S subunits apart.
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Eukaryotic Initiation 2
3 is the ternary complex of eIF2- GTP-Met-tRNAi
Met
eIF4 recognizes methylated 5-cap of mRNA, allows
binding of small subunit.
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Eukaryotic Initiation 3
It then proceeds in the 3 direction until it
hits the first AUG (usually).
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Euk and Pro initiation

• Although eukaryotic is more complex, there are
  important similarities.
• Both use GTP hydrolysis to provide energy to
  search for start codon.

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Elongation (a.a. addition)
In elongation, an incoming aminoacyl-tRNA moves
through three ribosomal sites Comes into A
site, pairing anticodon to codon. Ribosome
shifts it over to the about the distance of one
codon as it catalyzes the peptide
bond formation. tRNA without amino acid is
now ejected from the E site for reuse, and the A
site is ready for the next aminoacyl-tRNA.
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Termination
Two kinds of termination factors Recognize
STOP codons Hydrolyze peptidyl-tRNA bond
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Polyribosomes
Circular polyribosome has advantage of
efficiency. In prokaryotes only, the ribosome can
latch onto an mRNA before it has been completed,
following closely behind the RNA polymerase
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Antibiotic inhibition

• Puromycin Streptomyces alboniger similar to 3
  end aminoacyl-tRNA premature polypeptide
  synthesis termination
• Tetracycline blocking A site
• Chloramphenicol blocking peptidyl transfer
  bacterial, mitochondrial, chloroplast
• Cycloheximide blocking peptidyl transfer 80S
  eukaryotic ribosome
• Streptomycin misreading genetic code bacterial

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Toxin inhibition

• Diphteria toxin inactivate eEF2
• Ricin castor bean inactive 60S ribosome

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End
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Translational control and post-translational
events

• Translational control
• Polyproteins
• Protein targeting
• Protein modification
• Protein degradation

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Translational control

• In prokaryotes, the level of translation of
  different cistrons can be affected by (a) the
  binding of short antisense molecules,
• (b) the relative stability to nucleases of parts
  of the polycistronic mRNA ,
• (c) the binding of proteins that prevent
  ribosome access.

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• In eukaryotes,
• protein binding can also mask the mRNA and
  prevent translation,
• repeats of the sequence 5'-AUUUA -3' can make the
  mRNA unstable and less frequently translated.

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Polyprotein

• A single translation product that is cleaved to
  generate two or more separate proteins is called
  a polyprotein. Many viruses produce polyprotein.

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Protein targeting

• The ultimate cellular location of proteins is
  often determined by specific, relatively short
  amino acid sequence within the proteins
  themselves. These sequences can be responsible
  for proteins being secreted,
  imported into the nucleus or targeted to
  other organelles.

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Prokaryotic protein targeting secretion
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Eukaryotic protein targeting

• Targeting in eukaryotes is necessarily more
  complex due to the multitude of internal
  compartments
• There are two basic forms of targeting pathways

2.
1.
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The secretory pathwayin eukaryotes
(co-translational targeting)
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• The signal sequence of secreted proteins causes
  the translating ribosome to bind factors that
  make the ribosome dock with a membrane and
  transfer the protein through the membrane as it
  is synthesized. Usually the signal sequence is
  then cleaved off by signal peptidase.

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Protein modification

• Cleavage
• To remove signal peptide
• To release mature fragments from polyproteins
• To remove internal peptide as well as trimming
  both N-and C-termini

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• Covalent modification
• Acetylation
• Hydroxylation
• Phosphorylation
• Methylation
• Glycosylation
• Addition of nucleotides.

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• Phosphorylation

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Protein degradation

• Different proteins have very different
  half-lives. Regulatory proteins tend to turn over
  rapidly and cells must be able to dispose of
  faulty and damaged proteins.

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Protein degradation process

• Faulty and damaged proteins are attached to
  ubiquitins (ubiquitinylation).
• The ubiquitinylated protein is digested by a 26S
  protease complex (proteasome) in a reaction that
  requires ATP and releases intact ubiquitin for
  re-use.

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• In eukaryotes, it has been discovered that the
  N-terminal residue plays a critical role in
  inherent stability.
• 8 N-terminal aa correlate with stability
  Ala Cys Gly Met Pro Ser Thr Val
• 8 N-terminal aa correlate with short t1/2
  Arg His Ile Leu Lys Phe Trp Tyr
• 4 N-terminal aa destabilizing following chemical
  modification
  Asn Asp Gln Glu

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