Welcome Each of You to My Molecular Biology Class

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Welcome Each of You to My Molecular Biology Class
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Molecular Biology of the Gene, 5/E --- Watson et
al. (2004)
Part I Chemistry and Genetics Part II
Maintenance of the Genome Part III Expression
of the Genome Part IV Regulation Part V Methods
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Part III Expression of the Genome

• This part concerned with one of the greatest
  challenges in understanding the gene-how the gene
  is expressed.

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Part III Expression of the Genome
Ch 12 Mechanisms of transcription Ch 13 RNA
splicing Ch 14 Translation Ch 15 The genetic
code
translation
protein
DNA
RNA
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Chapter 14 Translation
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Translation extremely costs

• In rapid growing bacterial cells, protein
  synthesis consumes
• 80 of the cells energy
• 50 of the cells cells dry weight

Why?
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The main challenge of translation

• The genetic information in mRNA cannot be
  recognized by amino acids.
• The genetic code has to be recognized by an
  adaptor molecular (translator), and this adaptor
  has to accurately recruit the corresponding amino
  acid.

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Translation machinery

1. mRNAs (5 of total cellular RNA)
2. tRNAs (15)
3. aminoacyl-tRNA synthetases (??tRNA???)
4. ribosomes (100 proteins and 3-4 rRNAs--80)

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Outline

• Topics 1-4 Four components of translation
  machinery.
• T1-mRNA T2-tRNA T3-Attachment of amino acids
  to tRNA (aminoacyl-tRNA synthetases) T4-The
  ribosome
• Topic 5-6 Translation process.
• T5-initiation T6-elongation T7-termination.
• Topic 8 Translation-dependent regulation of mRNA
  and protein stability

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Topic 1 mRNA

• Only a portion of each mRNA can be translated.
• The protein-coding region of the mRNA consists of
  an ordered series of 3-nt-long units called
  codons that specify the order of amino acids.

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1-1 polypeptide chains are specified by ORF

• The protein coding region of each mRNA is
  composed of a contiguous, non-overlapping string
  of codons called an opening reading frame (ORF) .
• An ORF should begins with a start codon and end
  with stop codon.
• mRNA containing more than one ORF is called
  polycistronic mRNAs.

Message RNA
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Fig 14-1 Three possible reading frames of the E.
coli trp leader sequence
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1-2 Prokaryotic mRNAs have a ribosome binding
site that recruits the translational machinery
Message RNA
1-3 Eukaryotic mRNA are modified at their 5 and
3 ends to facilitate translation.
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• Ribosome binding site (RBS) or SD-sequence in
  prokaryotic mRNA, complementary with the sequence
  at the 3 end of 16S rRNA.

Fig 14-2-a structure of mRNA
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Once
Kozak sequence
Fig 14-2-b

• Eukaryotic mRNA uses a methylated cap to recruit
  the ribosome. Once bound, the ribosome scans the
  mRNA in a 5-3 direction to find the AUG start
  codon.
• Kozak sequence increases the translation
  efficiency.
• Poly-A in the 3 end promotes the efficient
  recycling of ribosomes

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Topic 2 tRNA
At the heart of protein synthesis is the
translation of nucleotide sequence information
into amino acids. This work is accomplished by
tRNA.
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2-1 tRNA are adaptors between codons and amino
acids

1. The are many types of tRNA molecules in cell
   (40).
2. Each tRNA molecule is attached to a specific
   amino acids (20) and each recognizes a particular
   codon, or codons (61), in the mRNA
3. All tRNAs end with the sequence 5-CCA-3 at the
   3 end, where the aminoacyl tRNA synthetase adds
   the amino acid.

TRANSFER RNA
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Primary structure

1. tRNAs are 75-95 nt in length.
2. There are 15 invariant and 8 semi-invariant
   residues. The position of invariant and
   semi-variant nucleosides play a role in either
   the secondary and tertiary structure.
3. There are many modified bases, which sometimes
   accounting for 20 of the total bases in one tRNA
   molecule. Over 50 different types of them have
   been observed.

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1. Pseudouridine (?U) is a modified base. These
   modified bases in tRNA lead to improved tRNA
   function

Fig 14-3 unusual bases
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2-2 tRNAs share a common secondary structure
that resemble a cloverleaf
TRANSFER RNA

• The cloverleaf structure is a common secondary
  structural representation of tRNA molecules which
  shows the base paring of various regions to form
  four stems (arms) and three loops.

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Fig 14-4 the secondary structure
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tRNA secondary structure
D loop
T loop
Anticodon loop
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Amino acid acceptor stem

• The 5-and 3-end are largely base-paired to form
  the amino acid acceptor stem which has no loop.

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D-arm and D-loop

• Composed of 3 or 4 bp stem and a loop called the
  D-loop (DHU-loop) usually containing the modified
  base dihydrouracil.

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Anticodon loop and anticodon loop

• Consisting of a 5 bp stem and a 7 residues loop
  in which the anticodon is located. The 3-nt
  anticodon sequence is used to recognize the codon
  sequence in the mRNA

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Variable arm and T-arm

• Variable arm 3 to 21 residues and may form a
  stem of up to 7 bp.
• T-arm is composed of a 5 bp stem ending in a
  loop containing the invariant residues GT?C.
  

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2-3 tRNAs have an L-shaped 3-D structure
TRANSFER RNA
Fig 14-5 the 3-D structure of tRNA
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• Formation
• 9 hydrogen bones (tertiary hydrogen bones)
  help the formation of tRNA tertiary structure,
  mainly involving in the base paring between the
  invariant bases.

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• Base pairing between residues in the D-and T-arms
  fold the tRNA molecule into an L-shape, with the
  anticodon loop at one end and the amino acid
  acceptor site at the other (Fig. 14-5). The base
  pairing is strengthened by base stacking
  interactions.

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Topic 3 attachment of amino acids to tRNA

• Amino acids should attach to tRNA first before
  adding to polypeptide chain.
• tRNA molecules to which an amino acid is attached
  are said to be charged, and tRNAs lacking an
  amino acid are said to uncharged.

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3-1 tRNAs are charged by attachment of an amino
acid to the 3 terminal A of the tRNA via a high
energy acyl linkage
ATTACHMENT OF AMINO ACIDS TO tRNA

• The energy released when the high-energy bond is
  broken helps drive the peptide bond formation
  during protein synthesis.

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3-2 Aminoacyl tRNA synthetases charge tRNA in two
steps
ATTACHMENT OF AMINO ACIDS TO tRNA

• 1. Adenylylation (????) of amino acids transfer
  of AMP to the COO- end of the amino acids.
• 2. tRNA charging transfer of the adenylylated
  amino acids to the 3 end of tRNA, generating
  aminoacyl-tRNAs.

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Also see Figure 14-6 in your text book

• Reaction step
• First, the aminoacyl-tRNA synthetase attaches AMP
  to the-COOH group of the amino acid utilizing ATP
  to create an aminoacyl (???) adenylate (???)
  intermediate.
• Then, the appropriate tRNA displaces the AMP.

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Proofreading

• Proofreading occurs at step 2 when a synthetase
  carries out step 1 of the aminoacylation reaction
  with the wrong, but chemically similar, amino
  acid.
• Synthetase will not attach the aminoacyl
  adenylate to the cognate tRNA, but hydrolyze the
  aminoacyl adenylate instead.

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3-3 each aminoacyl tRNA synthetase
attaches a single amino acids to one or more
tRNAs---accurate charging is essential
ATTACHMENT OF AMINO ACIDS TO tRNA

• Each of the 20 amino acids is attached to the
  appropriate tRNA (s) by aminoacyl-tRNA
  synthetases.
• Most amino acids are specified by more than one
  codon, and by more than one tRNA as well.

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• The same synthetase is responsible for charging
  all tRNAs for a particular amino acids.
• Consequently, most organisms have 20 synthetases
  for 20 different amino acids.

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tRNA charging by Aminoacyl-tRNA synthetases is
specific

• Nomenclature of tRNA-synthetases and charged tRNAs

Amino acid serine Cognate tRNA tRNASer Cognate
aminoacyl-tRNA synthetase Ser-tRNA
synthetase Aminoacyl-tRNA Ser-tRNASer
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There are two classes of tRNA synthetases.

• Class I attach the amino acids to the 2OH of
  the tRNA, and is usually monomeric.
• Class II attach the amino acids to the 3OH of
  the tRNA, and is usually dimeric or tetrameric

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3-4 tRNA synthetases recognize unique structure
features of cognate tRNAs
ATTACHMENT OF AMINO ACIDS TO tRNA

• The recognition has to ensure two levels of
  accuracy (1) each tRNA synthetase must recognize
  the correct set of tRNAs for a particular amino
  acids (2) each synthetase must charge all of
  these isoaccepting tRNAs (????synthetase??????tRNA
  s)

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• The specificity determinants for accurate
  recognition are clusters at two distinct sites
  the acceptor stem and the anti-codon loop.

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Fig 14-8
Fig 14-7
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3-5 Aminoacyl-tRNA formation is very accurate
selection of the correct amino acid
ATTACHMENT OF AMINO ACIDS TO tRNA

• The aminoacyl tRNA synthetases discriminate
  different amino acids according to different
  natures of their side-chain groups.
• Some enzymes have editing pocket to do
  proofreading by matching the wrong product and
  hydrolyzing it (3-6).

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Ribosome is responsible to place the charged
tRNAs onto mRNA through base pairing of the codon
in mRNA and anticodon in tRNA
ATTACHMENT OF AMINO ACIDS TO tRNA

• 3-7 Ribosomes is unable to discriminate between
  correctly or incorrectly charged tRNAs
  (??????????)

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Topic 4 the ribosome
Fig 14-17 two views of the ribosome
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4-1 the ribosome is composed of a large and a
small subunit

• The large subunit contains the peptidyl
  transferase center, which is responsible for the
  formation of peptide bonds.
• The small subunit interacting with mRNA contains
  the decoding center, in which charged tRNAs read
  or decode the codon units of the mRNA.

RIBOSOMES
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Fig 14-13 Ribosome
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4-2 the large and the small subunits
undergone association and dissociation during
each cycle of translation
RIBOSOMES
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• Ribosome cycles In cells, the small and large
  ribosome subunits associate with each other and
  the mRNA, translate it, and then dissociate after
  each round of translation. This sequence of
  association and dissociation is called the
  ribosome cycle.

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Fig 14-14 Overview of the events of
translation/ribosome cycle
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Polysome/polyribosome an mRNA bearing multiple
ribosomes

• Each mRNA can be translated simultaneously by
  multiple ribosomes

Fig 14-15 A polyribosome
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4-3 New amino acids are attached to the
C-terminus of the growing polypeptide chain.
RIBOSOMES
Protein is synthesized in a N- to C- terminal
direction
4-4 Peptide bonds are formed by transfer of the
growing peptide chain from peptidyl- tRNA to
aminoacyl-tRNA.
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Fig 14-16
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The structure of the ribosome

• 4-5 Ribosomal RNAs are both structural and
  catalytic determinants of the ribosomes
• 4-6 The ribosome has three binding site for tRNA.
• 4-7 Channels through the ribosome allow the mRNA
  and growing polypeptide to enter and/or exit the
  ribosome.

RIBOSOMES
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Fig 14-19 3-D structure of the ribosome including
3 bound tRNA
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Three binding site for tRNAs
Fig 14-18
A site to bind the aminoacylated-tRNA B-site to
bind the peptidyl-tRNA E-site to bind the
uncharged tRNA
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Channels for mRNA entering and exiting are
located in the small subunit (see Fig. 14-18)
There is a pronounced kink in the mRNA between
the two codons at P and A sites. This kink places
the vacant A site codon for aminoacyl-tRNA
interaction.
Fig 14-20
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Channel for polypeptide chain exiting locates in
the large subunit (see Fig. 14-18)
The size of the channel only allow a very limited
folding of the newly synthesized polypeptide
Fig 14-21
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• We now know that the rRNAs are not simply
  structural components of the ribosomes. Rather,
  they account for the key function of the
  ribosomes.
• Most ribosomal proteins are on the periphery of
  the ribosomes, not in its interior.
• So, its inferred that the contemporary ribosome
  evolved from a primitive protein synthesis
  machine that was composed entirely of RNA.

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Translation processT5 Initiation of
translationT6 Elongation of translationT7
termination of translation Watch the animation
on your study CD
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Questions

1. Compare the mechanism of translation initiation
   in prokaryotes and eukaryotes (similarity and
   difference)
2. How do prokaryotes and eukaryotes find the
   translation start sites?
3. How do aminoacyl-tRNA synthetases and the
   ribosomes contribute to the fidelity of
   translation, respectively?

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Overview of the events of translation
Fig 14-14
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5-6 Translation initiation factors hold
eukaryotic mRNAs in circles
INITIATION OF TRANSLATION
Try to explain how the mRNA poly-A tail
contributes to the translation efficiency?
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6-3 Ribosome is a ribozyme
ELONGATION OF TRANSLATION
Fig 14-33
Catalysis requires distance in the 1-3 Å, and
only RNA residues are present 18 Å from the
active site.
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EF-G mimics a tRNA molecule so as to displace the
tRNA bound to the A site
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Topic 8 translation dependent regulation of mRNA
and protein stability

• Here regulation refers cellular processes that
  deal with defective mRNA and their translated
  product.

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8-1 The SsrA RNA rescues (??) ribosomes that
translate broken mRNAs lacking a stop codon
(prokaryotes)

1. The ribosomes are trapped or stalled on the
   broken mRNA lacking a stop codon
2. The stalled ribosomes are rescued by the action
   of a chimeric RNA molecule that is part tRNA and
   part mRNA, called tmRNA.
3. SsrA is a 457-nt tmRNA

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Fig 14-39 SsrA rescues the stalled ribosomes
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8-2 Eukaryotic cells degrade mRNAs that are
incomplete or have premature stop codons
Translation is tightly linked to the process of
mRNA decay in eukaryotic cells
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Nonsense mediated mRNA decay
When an mRNA contains a premature stop codon
(nonsense codon), the mRNA is rapidly degraded by
nonsense mediated mRNA decay. Pre-releasing the
ribosome at the nonsense codon prior to reaching
the exon-junction complex initiates a talk
between the complex and ribosome to remove the 5
cap from the mRNA
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1. Translation of a normal eukaryotic mRNA
displace all the exon junction complex
Fig 14-40a
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2. Nonsense mediated mRNA decay
Fig 14-40b
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Nonstop mediated decay

• Non-stop mediated mRNA decay rescues ribosomes
  that translate mRNAs lacking a stop codon
• The lack of a stop codon results in ribosome
  translation into the poly-A tail to produce
  poly-Lys at the C-terminus of the polypeptide
  the poly-Lys marks the newly synthesized for
  rapid degradation.

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1. The ribosome eventually stalls at the 3 end of
   the mRNA, which is bound by the Ski7 protein that
   triggers the ribosome dissociation and recruits a
   3-5 exonuclease activity to degrade the
   nonstop mRNA.

Fig 14-40c
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Key points of the chapter

1. The main challenge of translation and the
   solution
2. The structure and function of four components of
   the translation machinery.
3. Translation initiation, elongation and
   termination (????????????)
4. The mRNA and protein stability dependent on
   translation (????????,?????)