Fundamentals of Cell Biology

Chapter 8: Protein Synthesis and Sorting

• 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 (1)

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.

• 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 converts the DNA genetic code into RNA

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'-to-3' 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; anti-terminator 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) co-

translationally, 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 ER-assisted 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 -actin mRNA