protein synthesis, processing, and...

Protein Synthesis, Processing, and Regulation

8 Protein Synthesis, Processing, and Regulation

Chapter Outline

• Translation of mRNA

• Protein Folding and Processing

• Regulation of Protein Function

• Protein Degradation

Introduction

Translation is the synthesis of proteins as directed by mRNA templates.

But it is only the first step in the formation of a functional protein.

The polypeptide chain must then fold into the appropriate conformation and often undergoes various processing steps.

Gene expression is regulated at the level of translation in both prokaryotic and eukaryotic cells.

There are also multiple controls on the amounts and activities of intracellular proteins, which ultimately regulate all aspects of cell behavior.

Translation of mRNA

Proteins are synthesized from mRNA templates by a process that has been highly conserved throughout evolution.

All mRNAs are read in the 5′ to 3′ direction, and polypeptide chains are synthesized from the amino to the carboxy terminus.

Each amino acid is specified by three bases (a codon) in the mRNA.

Translation is carried out on ribosomes, with tRNAs serving as adaptors.

Protein synthesis involves interactions between three types of RNA molecules (mRNA, tRNA, and rRNA), plus other proteins.

Translation of mRNAtRNAs align amino acids with corresponding codons on the

mRNA template.

They are 70 to 80 nucleotides long and have characteristic cloverleaf structures resulting from complementary base pairing between different regions.

Translation of mRNA

All tRNAs fold into compact L shapes, to fit onto ribosomes during translation.

All tRNAs have the sequence CCA at the 3′ terminus, and amino acids are covalently attached to the ribose of the terminal adenosine.

The anticodon loop binds to the appropriate codon by complementary base pairing.

Attachment of amino acids to specific tRNAs is mediated by enzymes called aminoacyl tRNA synthetases.

Each of these 20 enzymes recognizes a single amino acid, as well as the correct tRNA to attach it to.

The attachment occurs in two steps.

Translation of mRNA

1. The amino acid is joined to AMP, forming aminoacyl AMP.

2. The amino acid is transferred to the 3′ CCA terminus of the tRNA and AMP is released.

Translation of mRNAThe amino acid is then aligned on the mRNA template by

complementary base pairing.

At the third codon position, base pairing is relaxed, allowing G to pair with U, and inosine (I) in the anticodon to pair with U, C, or A.

(Guanosine is modified to inosine in the anticodons of some tRNAs.)

Figure 8.3 Nonstandard codon-anticodon base pairing (Part 2)

Translation of mRNA

Ribosomes are named according to sedimentation rates in ultracentrifugation: 70S for bacterial and 80S for eukaryotic.

Cells have many ribosomes, illustrating the importance of protein synthesis. E. coli has about 20,000; growing mammalian cells can have ten million.

All ribosomes have two subunits; each subunit contains both rRNA and proteins.

The subunits of eukaryotic ribosomes are larger and have more proteins than prokaryotic ribosomes.

Figure 8.4 Ribosome structure

Translation of mRNA

rRNAs form characteristic secondary structures by complementary base pairing.

Subsequent folding results in distinct 3-D structures.

Translation of mRNAIt was thought that ribosomal proteins catalyzed protein synthesis, but later

shown that rRNA has catalytic activity.

Noller and colleagues in 1992 showed that the large ribosomal subunit is able to catalyze formation of peptide bonds (the peptidyl transferase reaction) even after 90% of the ribosomal proteins have been removed.

Translation of mRNAUnambiguous evidence for rRNA catalysis came from high-resolution

structural analysis of the 50S ribosomal subunit, in 2000.

Ribosomal proteins are absent from the site of the peptidyl transferase reaction showing that rRNA was responsible for catalyzing peptide bond formation.

Translation of mRNA

It is now thought that ribosomal proteins play a largely structural role, and the large ribosomal subunit functions as a ribozyme.

This has evolutionary implications: RNAs are thought to have been the first self-replicating macromolecules.

The role of rRNA in the formation of peptide bonds extends the catalytic activities of RNA beyond self-replication to direct involvement in protein synthesis.

This may provide an important link for understanding the early evolution of cells.

Translation of mRNAProkaryotic and eukaryotic mRNAs contain untranslated regions (UTRs)

at the ends.

Eukaryotic mRNAs usually encode a single polypeptide chain.

Many prokaryotic mRNAs encode multiple polypeptides that are synthesized independently from distinct initiation sites (e.g., the lacoperon).

Translation of mRNA

Messenger RNAs that encode multiple polypeptides are called polycistronic.

Monocistronic mRNAs encode a single polypeptide.

In both prokaryotic and eukaryotic cells, translation always initiates with methionine, usually encoded by AUG.

The signals that identify initiation codons are different in prokaryotic and eukaryotic cells.

Translation of mRNA

Initiation codons in bacterial mRNAs are preceded by a Shine-Dalgarno sequence, that aligns the mRNA on the ribosome.

Thus they can initiate translation at the 5′ end of an mRNA and at internal initiation sites of polycistronic mRNAs.

Eukaryotic mRNAs are recognized by the 7-methylguanosine cap at the 5′ terminus.

The ribosomes then scan downstream of until they encounter the initiation codon.

Figure 8.8 Signals for translation initiation

Translation of mRNATranslation occurs in three stages: initiation, elongation, and termination.

A specific initiator methionyl tRNA and the mRNA bind to the small ribosomal subunit.

The large ribosomal unit then joins, forming a functional ribosome.

Translation of mRNA

Many nonribosomal proteins are also required for various stages of translation.

Translation of mRNA

In bacteria, initiation starts with a 30S ribosomal subunit bound to initiation factors IF1 and IF3.

Then the mRNA, the initiator N-formylmethionyl (fMet) tRNA, and IF2 (bound to GTP) join the complex.

IF1 and IF3 are released, a 50S subunit binds to the complex, and IF2 is released.

Figure 8.10 Initiation of translation in bacteria

Translation of mRNAIn eukaryotes initiation requires at least 11 proteins, designated eIFs

(eukaryotic initiation factors).

The initiator methionyl tRNA is bound to eIF2, and the mRNA is brought to the complex by eIF4E.

Figure 8.11 Initiation of translation in eukaryotic cells (Part 2)

Translation of mRNA

The mechanism of elongation in prokaryotic and eukaryotic cells is similar.

The ribosome has three binding sites: P (peptidyl), A (aminoacyl), and E (exit) sites.

The initiator methionyl tRNA is bound at the P site.

The next aminoacyl tRNA binds to the A site by pairing with the second codon of the mRNA.

An elongation factor (EF-Tu in prokaryotes, eEF1 in eukaryotes) complexed to GTP brings the aminoacyl tRNA to the complex.

Figure 8.12 Elongation stage of translation

Translation of mRNA

Selection of the correct aminoacyl tRNA determines the accuracy of protein synthesis.

Base pairing alone can’t account for the accuracy of protein synthesis.

A “decoding center” in the small ribosomal subunit recognizes correct codon-anticodon base pairs and discriminates against mismatches.

Insertion of a correct aminoacyl tRNA into the A site triggers a conformational change that induces hydrolysis of GTP bound to eEF1α and release of the elongation factor.

Then the peptide bond is formed, catalyzed by the large ribosomal subunit and the now uncharged initiator tRNA is at the P site.

Translation of mRNA

Then translocation occurs, during which the ribosome moves three nucleotides along the mRNA, positioning the next codon in an empty A site.

This step translocates the peptidyl tRNA from A to P, and the uncharged tRNA from P to E.

A new aminoacyl tRNA binds to the A site and induces release of the uncharged tRNA from the E site.

Translation of mRNAAs elongation continues, the eEF1α (or EF-Tu) released from the ribosome bound to GDP

must be reconverted to its GTP form.

This requires a third elongation factor, eEF1βγ (EF-Ts in prokaryotes).

Regulation of eEF1α by GTP binding and hydrolysis represents a common method of protein regulation.

Translation of mRNAElongation continues until a stop codon (UAA, UAG, or UGA) is translocated into the

A site.

Release factors recognize the signals and terminate protein synthesis.

In prokaryotic cells RF1 recognizes UAA or UAG, RF2 recognizes UAA or UGA.

In eukaryotic cells eRF1 recognizes all three stop codons.

Translation of mRNAmRNAs can be translated simultaneously by several ribosomes.

Once a ribosome has moved away from the initiation site, another can bind to the mRNA and begin synthesizing.

The group of ribosomes bound to an mRNA molecule is called a polyribosome, or polysome.

Translation of mRNA

Regulation of translation also plays a key role in modulating gene expression.

Regulation includes translational repressor proteins and noncoding microRNAs.

Global translational activity of cells is modulated in response to cell stress, nutrient availability, and growth factor stimulation.

Translation of ferritin (a protein that stores iron) mRNA is regulated by repressor proteins.

When iron is absent, iron regulatory protein (IRP) binds to a the iron response element (IRE) in the 5′ UTR, blocking translation.

Figure 8.16 Translational regulation of ferritin

Translation of mRNASome translational repressors bind to specific sequences in the 3′ UTR.

Some bind to initiation factor eIF4E, interfering with its interaction with eIF4G and inhibiting initiation of translation.

Translation of mRNAProteins that bind to the 3′ UTRs are also responsible for localizing mRNAs

to specific regions of cells.

Localization of mRNAs to specific regions of eggs or embryos plays an important role in development, allowing proteins to be synthesized at appropriate sites.

Translation of mRNA

Translational regulation is particularly important during early development.

Many mRNAs with short poly-A tails are stored in oocytes; translation is activated at fertilization or later stages of development.

Lengthening the poly-A tails allows binding of poly-A binding protein (PABP), which stimulates translation.

Translation of mRNA

RNA interference (RNAi) mediated by short double-stranded RNAs is used as an experimental tool to block gene expression at the level of translation.

The RNA interference pathway inhibits translation and/or induces degradation of target mRNAs.

Two types of small RNAs mediate RNA interference:

Small interfering RNAs (siRNAs)—produced from double-stranded RNAs by the nuclease Dicer.

MicroRNAs (miRNAs)—transcribed by RNA polymerase II, then cleaved by nucleases Drosha and Dicer.

Translation of mRNAOne strand is incorporated into the RNA-induced silencing complex (RISC).

siRNAs or miRNAs that pair perfectly can induce cleavage of the targeted mRNA.

Most miRNAs form mismatches that repress translation.

Figure 8.19 Regulation of translation by miRNAs (Part 2)

Translation of mRNAAs many as 1000 miRNAs are encoded in mammalian

genomes; each can target up to 100 different mRNAs.

At least one-third of protein-coding genes may be regulated by miRNAs.

They are important in embryonic development, and may contribute to heart disease and cancer.

Translation can also be regulated by modification of initiation factors.

This results in global effects on overall translational activity rather than translation of specific mRNAs.

Phosphorylation of eIF2 and eIF2B by regulatory protein kinases blocks exchange of bound GDP for GTP, inhibiting initiation of translation.

Figure 8.20 Regulation of translation by phosphorylation of eIF2 and eIF2B (Part 1)

Translation of mRNARegulation of eIF4E: growth factors activate protein kinases that

phosphorylate regulatory proteins (eIF4E binding proteins, or 4E-BPs).

In the absence of growth factors, the nonphosphorylated 4E-BPs bind to eIF4E and inhibit translation.

Protein Folding and Processing

Polypeptide chains must undergo folding and other modifications to become functional proteins.

The 3-D conformations result from interactions between the side chains of the amino acids.

All information for the correct conformation is provided by the amino acid sequence.

Chaperones are proteins that facilitate the folding of other proteins.

They act as catalysts that facilitate assembly without becoming part of the assembled complex.

They bind to and stabilize unfolded or partially folded polypeptides that are intermediates leading to the final correctly folded state.

Chaperones bind to nascent polypeptide chains that are still being translated on ribosomes.

The chain must be protected from aberrant folding or aggregation with other proteins until synthesis of an entire domain is complete.

Figure 8.22 Action of chaperones during translation

Protein Folding and ProcessingChaperones also stabilize unfolded polypeptide chains during their transport into

organelles.

Example: partially unfolded proteins stabilized by chaperones are transported across the mitochondrial membrane.

Chaperones in the mitochondrion then facilitate subsequent folding.


Many chaperones were initially identified as heat-shock proteins (Hsp)—expressed in cells that were subjected to high temperatures.

Hsp stabilize and facilitate refolding of proteins that have been partially denatured.

Hsp70 chaperones and chaperonins are found in both prokaryotic and eukaryotic cells.

Hsp70 proteins stabilize unfolded polypeptide chains during translation by binding to short hydrophobic segments.

Protein Folding and ProcessingThe unfolded polypeptide is then transferred to a chaperonin, where folding

takes place.

Chaperonins consist of protein subunits arranged in two stacked rings to form a double-chambered structure. This isolates the protein from the cytosol and other unfolded proteins.


Two enzymes act as chaperones by catalyzing protein folding: Protein disulfide isomerase (PDI) catalyzes disulfide bond

formation. PDI is one of the most abundant proteins in the ER, where an

oxidizing environment allows (S—S) linkages.

Protein Folding and Processing• Peptidyl prolyl isomerase catalyzes isomerization of peptide bonds that

involve proline residues.

Isomerization between the cis and trans configurations of prolyl-peptide bonds, could otherwise be a rate-limiting step in protein folding.

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