Fundamentals of Cell Biology
Chapter 8: Protein Synthesis and
Sorting
Chapter Summary: The Big Picture (1)
• Chapter foci:
- Transcription events from unwinding of the DNA to
the production of functional RNA
- Translation events which govern RNA to protein
conversion
- Protein sorting events which send newly synthesized
proteins to the correct location in cell
Chapter Summary: The Big Picture (2)
• Section topics:
– Transcription converts the DNA genetic code
into RNA
– Proteins are synthesized by ribosomes using
an mRNA template
– At least five different mechanisms are
required for proper targeting of proteins in a
eukaryotic cell
Transcription converts the DNA genetic
code into RNA
• Key Concepts (1):
– Transcription resembles DNA replication, in that
DNA is separated into a “bubble” of single strands,
and the single-stranded DNA serves as a template.
– Transcription differs from DNA replication, in that
typically only one side of the transcription bubble is
used as a template, and the bubble does not grow
in size as transcription progresses.
– The steps of transcription are grouped into three
stages, called initiation, elongation, and termination.
Transcription converts the DNA genetic
code into RNA
• Key Concepts (2):
– Eukaryotic RNAs undergo posttranscriptional
processing; mRNAs are the most studied forms of
processed RNA.
– Following processing, RNAs are bound to several
proteins and transported into the cytosol through
the nuclear pore.
Transcription
RNA polymerases transcribe genes in a
"bubble" of single-stranded DNA
• Transcription bubble
– Similar to
replication bubble
– Unidirectional
– Single strand
Figure 08.01: An
overview of the
transcription bubble.
Transcription occurs in three stages (1)
• In eukaryotes, three different RNA polymerases
are used to transcribe different forms of RNA
– RNA polymerase I, II, II (pol I, II, III)
• Transcription begins after a RNA polymerase
binds to a promoter site on DNA
– Site of initiation by transcription factor
Transcription occurs in three stages (2)
• Transcription factors
form basal
transcription complex
Transcription: Initiation
• Transcription
startpoint – core
promotor
– TATA box
Figure 08.02:
Transcription has three
stages. Initiation, Step
Elongation, and
Termination.
Figure 08.03: Assembly of
the initiation complex for
DNA polymerase II.
Transcription: Elongation
• RNA transcript is
extended in the 5'-to3' direction as the
RNA polymerase
reads the template
DNA strand in the 3'to-5' direction
• Gyrase
• Topoisomerase
Figure 08.05:
The entire
transcription
bubble is
enclosed by
RNA
polymerase.
Figure 08.06: Topoisomerase induces positive
supercoiling of DNA.
Transcription: Termination
• termination is encoded by specific DNA
sequences called terminators
• some termination requires additional proteins to
bind to RNA polymerase which detect
sequences in the transcribed gene and induce
the polymerase to stop transcription
• terminators are not universally effective; antiterminator proteins can bind to the terminator
and suppress transcription resulting in
readthrough (polycistronic RNA)
In eukaryotes, messenger RNAs
undergo processing prior to leaving the
nucleus
• spliceosome controls
RNA splicing
• 5' and 3' ends of
messenger RNAs are
modified prior to
export
Figure 08.07:
Eukaryotic mRNA
is modified,
processed, and
transported.
Figure 08.08: 5'
modification occurs
before splicing and 3'
modification in the
nucleus.
RNA modifications
• 5’ methylguanosine
cap
• poly(A) tail
Figure 08.09: Eukaryotic mRNA has a methylated 5' cap. The cap protects
the 5' end of mRNA from nucleases and may be methylated at several
positions.
Figure 08.10: Exposure of the polyadenylation sequence by endonuclease and
exonuclease cleavage triggers addition of the poly(A) tail by pol(A) polymerase.
RNA export is unidirectional and
mediated by nuclear transport proteins
• poly(A)-binding
protein
• heterogeneous
nuclear
ribonucleoprotein
particle (hnRNP)
• messenger
ribonucleoprotein
particle (mRNP)
Figure 08.11: Initiation
of translation in
eukaryotes.
Figure 08.12: Some
portions of the hnRNP
are recycled into the
nucleus after passing
through the nuclear
pore. Others remain
and impact translation.
Proteins are synthesized by ribosomes using
an mRNA template
• Key Concepts:
– Translation is the term used to describe the
conversion of mRNA information into polypeptides.
– Translation requires cooperation between ribosomal
RNAs, transfer RNAs, messenger RNAs, and
numerous proteins.
– Translation is performed by one of the largest
molecular complexes in cells, the ribosome.
– The steps of translation are grouped into three
stages: initiation, elongation, and termination. These
are very different from the identically named stages of
transcription.
Translation occurs in three stages
• Key players
– Ribosome
– tRNAs
– Translation factors
• The ribosome has
three tRNA-binding
sites
– A, P, and E sites
Figure 08.13: The relative sizes of components of
the cellular translation machinery.
Figure 08.14: Cartoon depicting the structure of an
intact ribosome coupled to an mRNA.
Stage 1: Initiation requires base pairing
between mRNA and rRNA
• Goal = bring all of the elements necessary for
translation together into a giant cluster
• Ribosomal subunit to find ribosomal binding site
= Shine-Delgarno sequence = initiation site
• Once the mRNA and small subunit are properly
aligned, the first tRNA (initiator tRNA) binds to
the AUG, and the large ribosomal subunit
clamps down on the small subunit, forming an
intact ribosome
Stage 2: Elongation
• amino acid is added
to the carboxy
terminus of the
polypeptide in the A
site
Figure 08.15: The elongation cycle during translation.
Stage 3: Termination
• occurs when the bond
holding the
polypeptide to tRNA is
hydrolyzed
• Stop codon
• Release factors
Figure 08.15: The elongation cycle during translation.
5 different mechanisms are required for
proper targeting of proteins
• Key Concepts (1):
– Virtually all protein synthesis is centralized in the
cytosol for eukaryotic cells, and many of these
proteins are targeted to specific cellular locations by
signal sequences.
– Proteins that enter and leave the nucleus are
maintained in a functional shape at all times.
– Proteins enter the peroxisome in a functional, folded
state, but this transport is unidirectional.
Peroxisomal proteins appear to originate from
several sources, including the cytosol.
5 different mechanisms are required for
proper targeting of proteins
• Key Concepts (2):
– Proteins enter the endoplasmic reticulum (ER) cotranslationally, and are folded into their final shape
as they enter the ER lumen. They also undergo
extensive posttranslational modification.
– Distinct hydrophobic sequences in transmembrane
polypeptides are responsible for stabilizing them in
membranes.
5 different mechanisms are required for
proper targeting of proteins
• Key Concepts (3):
– Proteins enter mitochondria and chloroplasts through
very similar posttranslational mechanisms, suggesting
they share a common (prokaryotic) origin. Chaperone
proteins in the cytosol and interior of these organelles
help maintain these proteins in an unfolded and folded
state, respectively.
– Some mRNAs can be localized to specific regions of
the cytosol, thereby controlling where the resulting
proteins are concentrated. The actin and microtubule
cytoskeletal networks assist in this.
Signal sequences code for proper
targeting of proteins
Figure 08.16: An overview of protein
targeting in eukaryotic cells. Note
that signal sequences lead the
insertion into most target organelles.
The nuclear import/export system
regulates traffic through nuclear pores
• Proteins transported in/out
of nucleus in folded,
functional state
• Nuclear localization
sequences (NLS) and
nuclear export signals
(NES) are amino acid
sequences recognized by
NLS and NES receptors
• Direction of nuclear
transport is controlled by
Ran
Figure 08.17: An overview of protein
transport into and out of the nucleus.
Proteins targeted to the peroxisome contain
peroxisomal targeting signals (PTS)
• Proteins are
transported into the
peroxisomal matrix in
their properly folded,
functional state
• PTS receptors return
to cytoplasm after
delivering cargo
• Import process not
well understood
Figure 08.18: A generalized model of
peroxisomal protein import.
Secreted proteins and proteins targeted to
the endomembrane system contain an ER
signal sequence
• Co-translational
• Unfolded
• Unidirectional
Figure 08.19: An overview of protein import in the
endoplasmic reticulum.
SRP – Translocon – Signal Peptidase
Figure 08.20: The four
classes of transmembrane
proteins, according to the
Singer classification system.
Figure 08.21: Integrating a Type I transmembrane protein with a
signal sequence in a single transmembrane domain.
Transmembrane proteins contain signal
anchor sequences
• Transmembrane
domain
• Signal anchor
sequence
• 4 types of
transmembrane
proteins: I-IV
Figure 08.22: Integrating membrane
proteins with a signal anchor sequence.
Transmembrane protein orientations
Figure 08.23: A model for the integration of multi-spanning membrane proteins.
Figure 08.24: N-linked glycosylation of
polypeptides in the ER.
Figure 08.25: GPI synthesis and modification of proteins.
As proteins enter the ER lumen, they
may be post-translationally modified
Figure 08.26: A disulfide bond in PDI is used to form
one in the nascent polypeptide.
Figure 08.27: BiP binds to exposed hydrophobic
patches in recently translocated proteins.
Figure 08.28: Chloroplast
proteins must cross two
membranes to enter the
stroma.
Chaperone proteins assist in the proper
folding of ER proteins
Figure 08.29: A model for mRNA can be transported by the cytoskeleton.
Terminally misfolded proteins in the ER
are degraded in the cytosol
• Misfolded polypeptides in ER are "reverse
translocated" back into cytosol
• Once incytosol, misfolded polypeptides are
ubiquitinated and subsequently degraded by
proteosomes
• Process of identifying, reverse translocating, and
destroying these polypeptides is called ERassisted degradation (ERAD)
Cytosolic proteins targeted to
mitochondria or chloroplasts contain an
N-terminal signal sequence
The cytoskeleton immobilizes and
transports mRNAs
• Example – zipcode
sequence in b-actin
mRNA