Essential Cell Biology

Third Edition

Chapter 7 (Continued) From DNA to Protein: How Cells Read the Genome
Copyright Garland Science 2010

From DNA to Protein: How Cells Read the Genome

The flow of g genetic information in all living g cells is DNA RNA p protein. This process, including transcription and translation, is called gene expression. Transcription is catalyzed by the enzyme RNA polymerase. Nucleotide q in the DNA determine where to start and stop p transcription. p sequences RNA differs from DNA by containing the sugar ribose instead of deoxyribose and the base uracil (U) instead of thymine (T). RNAs are synthesized as singlep into three-dimensional shapes p . stranded molecules and fold up Cells make several different functional types of RNAs, including messenger RNA (mRNA), which carries the instructions for making proteins; ribosomal RNA (rRNA) ), which is a component p of ribosomes; and transfer RNA (tRNA) ), which acts as an adaptor molecule in protein synthesis. To initiate transcription, eucaryotic RNA polymerases require the assembly of a complex p of general g transcription p factors at the p promoter, whereas bacterial RNA polymerase requires only an additional subunit, called sigma factor. In eucaryotic DNA, most genes are composed of a number of smaller coding g (exons) ( ) interspersed p with noncoding g regions g (introns). ( ) When a regions eucaryotic gene is transcribed, both the exons and introns are copied. Introns are removed from RNA in the nucleus by the process of RNA splicing.

Special nucleotide sequences signal the beginning and the end of an intron
Only O l the th nucleotide l tid sequences required i d to t remove an intron, i t are shown h below. b l The special sequences are recognized by small nuclear ribonucleoprotein particles (snRNPs), which contain small nuclear RNAs (snRNAs) and proteins. The Th snRNPs RNP form f the th core of f the th spliceosome li , which hi h carries i out t RNA splicing. li i The spliceosome mediates the cleavage of the RNA at the intron-exon borders and the covalent link of the exons. R stands for either A or G; Y stands for either C or U; N stands for any an nucleotide. n cleotide

An intron forms a branched structure during splicing

In the first step, p, the branch point adenine (A) in the intron sequence attacks the 5 splice site and cuts the sugar phosphate backbone of the RNA at this point. The cut 5 end of the intron becomes covalently linked to the 2-OH group of the ribose of the A to form a branched structure. The free 3-OH end of the exon sequence then reacts with the start of the next exon sequence, joining the two exons together into a continuous coding sequence and releasing the intron in the form of a lariat structure, which is eventually degraded.

Movie: RNA Splicing Mechanism

The -tropomyosin gene can be spliced in different ways

-tropomyosin is a protein that regulates contraction in muscle cells. Its transcript can be spliced in different ways to produce distinct mRNAs. This process is called alternative splicing, which allows many different proteins to be produced from the same gene. Some of the splicing patterns are specific for certain types of cells. The red arrowheads represent sites where poly-A addition can occur.

RNA-binding proteins signal that a mature mRNA is ready for export to the cytoplasm
As indicated on the left, the cap and poly-A tail of a mature mRNA are marked by proteins that recognize these modifications. In addition, a group of proteins called the exon junction complex (EJC) is deposited on the mRNA after successful RNA splicing has occurred. Once the mRNA is deemed export ready a nuclear transport receptor associates with it and guides it through the nuclear pore. Once in the cytosol, the mRNA can shed previously bound proteins and acquire new ones.

Procaryotes and eucaryotes handle their RNA transcripts differently

(A) In eucaryotic cells, the initial RNA contains both intron and exon sequences. sequences Its two ends are modified, and the introns are removed by RNA splicing. The resulting mRNA is then transported from the nucleus to the c toplasm where cytoplasm, here it is translated into protein. protein In reality these steps occur simultaneously. For example, the RNA cap is usually added and splicing often begins before the transcript has been completed. (B) In procaryotes, the production of mRNA molecules is simpler. The 5 end of an mRNA is produced by the initiation of transcription by RNA polymerase, and the 3 end is produced by the termination of transcription. Because procaryotic cells lack a nucleus, transcription and translation take place in a common compartment. Translation of a bacterial mRNA can therefore begin before its synthesis has been completed. The amount of protein in a cell depends on the efficiency of each of these steps and on the rates of degradation of the RNA and protein molecules.

(II) F From RNA t to P Protein t i

The nucleotide sequence of an mRNA is translated into the amino acid sequence of a protein via the genetic code
Each group of three consecutive nucleotides in RNA is called a codon and each specifies one amino acid. Because RNA is a linear polymer made of four different nucleotides, there are thus 4 x 4 x 4 = 64 combinations of three nucleotides: AAA, AAA AAC AAC, AAG AAG, and so on on. By convention, codons are always written with the 5-terminal nucleotide to the left. Most amino acids are represented by more than one codon. Codons for the same amino acid tend to contain the same nucleotides at the first and second positions and to vary at the third position. There are three codons (UAA, UAG, and UGA) that do not specify any amino acid but act as stop codons, signaling the end of the protein-coding sequence. One codon -AUG- acts both as an initiation codon, signaling the start of a proteincoding message, and as the codon that specifies methionine.

An RNA molecule can be translated in three possible reading frames

In the process of translating a nucleotide sequence (blue) into an amino acid sequence (red), the sequence of nucleotides in an mRNA molecule is read from the 5 to the 3 end in seq sequential ential sets of three nucleotides. In principle, the same RNA sequence can specify three completely different amino acid sequences, depending on the reading g frame. In reality, however, only one of these reading frames g encodes the actual message.

tRNA molecules are molecular adaptors, linking amino acids to codons

The cloverleaf structure of tRNA for phenylalanine with the base-pairing that creates the double-helical regions g and the anticodon that base-pairs p with a codon in mRNA ( (A). ) The amino acid matching the codon/anticodon pair is attached at 3 end of the tRNA. Views of the actual L-shaped tRNA, rotated 90o (B and C). The linear nucleotide sequence of the molecule (D). Schematic representation of tRNA, emphasizing the anticodon (E).

The genetic code is translated by means of two adaptors

The first adaptor is the aminoacyl-tRNA synthetase, which couples a particular amino acid to its corresponding tRNA; this coupling process is called charging. The second adaptor is the tRNA molecule itself, whose anticodon forms base pairs with the appropriate codon on the mRNA. A tRNA coupled with its amino acid is also called a charged tRNA. An error in either step - the charging or the binding of the charged tRNA to its codonwill cause the wrong amino acid to be incorporated into a protein chain. The amino acid tryptophan (Trp) is selected by the codon UGG on the mRNA.

Movie: tRNA

Ribosomes are found in the cytoplasm of a eucaryotic cell

This electron micrograph shows a thin section of a small region of cytoplasm. The ribosomes appear as black dots (red arrows). Some are free in the cytosol; others are attached to membranes of the endoplasmic reticulum.

A ribosome is a large complex of four RNAs and more than 80 proteins

Shown here are the components of eucaryotic ribosomes. Procaryotic ribosomes are very similar. similar Although ribosomal proteins greatly outnumber t b ribosomal ib l RNAs, the RNAs account for more than half the mass of the ribosome.

A ribosome has a binding site for mRNA and three binding sites for tRNA
The tRNA sites are designated the A-, P-, and E-sites (short for aminoacyl-tRNA, peptidyl-tRNA, and exit, respectively). (A) Three-dimensional structure of a bacterial ribosome with the small subunit in dark green and the large subunit in light green. tRNAs are shown bound in the E-site (red), the P-site P site (orange) (orange), and the A-site A site (yellow) (yellow). Although all three tRNA sites are shown occupied here, during protein synthesis only two sites are occupied at any one time. (B) Structure of the large (left) and small (right) ribosomal subunits arranged as though the ribosome in (A) were opened like a book. (C) ) Structure of the ( ribosome in (A) viewed from the top. (D) A ribosome (in the same orientation as C) C).

Translation takes place in a four-step cycle

Step 1, a tRNA carrying the next amino acid binds to the vacant A-site on the ribosome by forming g base p pairs with the codon that is exposed p there. Step 2, the carboxyl end of the polypeptide chain is uncoupled from the tRNA at the P-site and joined by a peptide bond to the amino group of the amino acid linked to the tRNA at the A-site. This reaction is catalyzed by the RNA-based peptidyl transferase, 23S rRNA, in the large subunit. The 23 rRNA is a ribozyme. Step 3, a shift of the large subunit relative to the small subunit moves the two tRNAs into the E- and P-sites of the large subunit. Step 4, the small subunit moves exactly three nucleotides along the mRNA molecule molecule, bringing it back to its original position relative to the large subunit. This movement resets the ribosome with an empty Asite so that the next aminoacyltRNA molecule can bind.

Ribosomal RNAs give the ribosome its overall shape

Structures of the two rRNAs - the 23S rRNA (blue) and the 5S rRNA (purple) - that form the core of the large subunit of a bacterial ribosome. One of the protein s b nits of the ribosome (L1) subunits is included as a reference point, as this protein forms a characteristic p protrusion on the ribosome surface. Ribosomal components are commonly designated by their S values l , which hi h refer f t to th their i rate of sedimentation in an ultracentrifuge.

Translation initiation in eucaryotes

In eucaryotes, the initiator tRNA, coupled to methionine, is first loaded into a small ribosomal subunit, along with additional proteins called translation initiation factors. The Th i initiator iti t tRNA is i di distinct ti t f from th the tRNA that th t normally carries methionine. Of all the charged tRNAs in the cell, only the charged initiator tRNA is capable of binding tightly to the P-site of the small ribosomal subunit. The loaded ribosomal subunit binds to the 5 end of an mRNA molecule, which is signaled by the h cap that h i is present on eucaryotic i mRNA. RNA The small ribosomal subunit then moves forward (5 to 3) along the mRNA searching for the first AUG. When this AUG is encountered, encountered several initiation factors dissociate from the small ribosomal subunit to make way for the large ribosomal subunit to assemble and complete the ribosome. Because the initiator tRNA is bound to the P-site, protein synthesis is ready to begin with the addition of the next charged tRNA to the A-site.

Movie: Translation

A single procaryotic mRNA molecule can p encode several different proteins.

In procaryotes, genes directing the different steps in a process are often organized into clusters (operons) that are transcribed together into a single mRNA. Unlike eucaryotic ribosomes, which recognize a 5 5 cap, procaryotic ribosomes initiate transcription at ribosome-binding sites (red), which can be located in the interior of an mRNA molecule. This feature enables procaryotes to synthesize several separate proteins from a single mRNA molecule.

Translation termination in eucaryotes

In the final phase of protein synthesis, the binding of release factor to an A-site bearing a stop codon terminates translation. The end of the protein-coding message in both procaryotes and eucaryotes is signaled by the presence of one of several codons called stop codons UAA, UAG, and UGA. The stop codons are not recognized by a tRNA and do not specify an amino acid, but instead signal to the ribosome to stop translation. The release factors bind to a stop codon that reaches the A-site on the ribosome, which alters the activity of the peptidyl transferase in the ribosome, causing it to catalyze the addition of a water instead of an amino acid to the peptidyltRNA. This reaction frees the carboxyl end of the polypeptide chain from its attachment to a tRNA molecule, and the completed protein chain is immediately released into the cytosol. The ribosome releases the mRNA and dissociates into its two separate subunits, which can then assemble on another mRNA molecule to begin a new round of protein synthesis.

Proteins are translated by polyribosomes

A series of ribosomes can simultaneously translate the same mRNA molecule. Electron micrograph of a polyribosome from a eucaryotic cell cell.

Antibiotics that inhibit protein or RNA synthesis

Many of our most effective antibiotics are compounds that act by inhibiting bacterial, but not eucaryotic protein or RNA synthesis. Some of these drugs exploit the small structural and functional differences between bacterial and eucaryotic ribosomes to interfere preferentially with bacterial protein synthesis. These compounds can thus be taken in high doses without being toxic to humans.

The Ubiquitin-Proteasome Pathway (UPP) mediates protein degradation in eukaryotes

The short-lived and misfolded proteins are recognized and degraded by the Ubiquitin-Proteasome Pathway (UPP). The enzymes (E1, E2, and E3) covalently attached ubiquitin to such proteins. The subsequent degradation of the ubiquitin-tagged protein by the 26S proteasome (composed of the catalytic 20S core and the 19S regulator). More recently, it has become evident that protein modification by ubiquitin also has unconventional (non-degradative) functions such as the regulation of DNA repair and endocytosis. These non-traditional functions are dictated by the number of ubiquitin units attached to proteins (mono- versus polyubiquitination) and also by the types of ubiquitin q chain linkage g that is p present. The deubiquitinating enzymes (DUBs) remove ubiquitin from its substrates.

Aaron Ciechanover

Avram Hershko

Irwin Rose

The Nobel Prize in Chemistry 2004 "for the discovery of ubiquitin-mediated protein degradation".

Protein production in a eucaryotic cell requires many steps

The final concentration of each protein depends on the efficiency of each step depicted. Even after a protein has been translated, its concentration can be regulated by degradation, and its activity regulated by the binding of small molecules and other post posttranslational modifications.

Many proteins require additional modification to become fully functional

To be useful to the cell, a completed polypeptide must fold correctly into its three threedimensional conformation, bind any cofactors required, and assemble with any protein partners. Noncovalent bond formation drives these changes. Many proteins also require covalent modification to become active. Although phosphorylation and glycosylation are the most common, more than 100 different types of covalent modifications of proteins are known known.