Chapter 17 Continued: From Gene to Protein

*Accurate translation requires two steps...*

(1) correct match between tRNA and amino acid, done by enzyme *aminoacyl-tRNA synthetase* (2) correct match between tRNA anticodon and mRNA codon, which is facilitated by the ribosome

*Shared Functions of 5' Cap and Poly-A Tail*

(1) facilitate export of mature mRNA from nucleus (2) help protect mRNA from degradation by hydrolytic enzymes (3) help ribosomes attach to 5' end of mRNA once mRNA reaches cytoplasm

*Three stages of translation...*

(1) initiation (2) elongation (3) termination • all three stages require protein "factors" that aid in the translation process *go to other card set for more info*

Figure 17.17 Schematic model with mRNA and tRNA

A tRNA fits into a binding site when its anticodon base-pairs with an mRNA codon. The P site holds the tRNA attached to the growing polypeptide. The A site holds the tRNA carrying the next amino acid to be added to the polypeptide chain. Discharged tRNA leaves from the E site.

Mutagens

physical or chemical agents that can cause mutations

Some introns contain sequences that may...

regulate gene expression

Nucleotide-Pair Substitution

replacement of one nucleotide and its partner with another pair of nucleotides

*Signal Peptide*

sequence of about 20 amino acids at or near the leading end of the polypeptide that marks polypeptides of proteins destined for the ER or for secretion and thus targets the protein to the ER

*snRNPs*

• small nuclear ribonucleoproteins • particles that recognize splice sites (short nucleotide sequence at each end of intron) • located in nucleus and composed of RNA and protein molecules • RNA in a snRNP is called a small nuclear RNA or *snRNA*

*RNA Splicing*

• stage of RNA processing that involves the removal of portions of the introns and the joining together of the exons • creates mRNA molecule with continuous coding sequence

*Missense Mutation*

• still code for an amino acid, but not the correct amino acid • substitutions that change one amino acid to another one • may have little effect on the protein

*Free Ribosomes*

• suspended in cytosol • synthesize proteins that stay and function in cytosol

Introns

• the noncoding segments of nucleic acid that lie between coding regions • called *in*tervening sequences

*Alternative RNA Splicing*

• type of eukaryotic gene regulation • some genes can encode more than one kind of polypeptide, depending on which segments are treated as exons during splicing, which is called alternative RNA splicing • consequently, the number of different proteins an organism can produce is much greater than its number of genes

Figure 17.10 RNA processing: Addition of 5' cap and poly-A tail

(1) a modified guanine nucleotide added to 5' end (2) 50-250 adenine nucleotides added to 3' end • enzymes modify two ends of eukaryotic pre-mRNA molecule; modified ends may promote export of mRNA from nucleus and help protect mRNA from degradation • when mRNA reaches cytoplasm, modified ends, in conjunction with certain cytoplasmic proteins, facilitate ribosome attachment • 5' cap and poly-A tail are not translated into protein, nor are regions called 5' untranslated region (5' UTR) and 3' UTR

Three properties of RNA enable it to function as an enzyme...

(1) it can form a three-dimensional structure because of its ability to base-pair with itself (can do this b/c RNA is single-stranded) (2) some bases in RNA contain functional groups that may participate in catalysis (3) RNA may hydrogen-bond with other nucleic acid molecules, which adds specificity to its catalytic activity

*Point mutations within a gene can be divided into two general categories...*

(1) nucleotide-pair substitutions (2) one or more nucleotide-pair insertions or deletions

Figure 17.15B Structure of tRNA

(b) *three-dimensional structure* and (c) *symbol used in book* • tRNA actually twists and folds into compact three-dimensional structure that is roughly L-shaped • loop extending from one end of L includes anticodon, the particular nucleotide triplet that base-pairs to specific mRNA codon • from other end of L-shaped tRNA protrudes its 3' end which is attachment site for an amino acid

Figure 17.15A Structure of tRNA

*Two-dimensional structure* • four base-paired regions and three loops are characteristic of all tRNAs, as is base sequence of amino acid attachment site at 3' end • anticodon triplet is unique to each tRNA type, as are some sequences in other two loops • asterisks mark bases that have been chemically modified, a characteristic of tRNA

*1 Nucleotide-Pair Deletion*

*frameshift* causing extensive missense

*1 Nucleotide-Pair Insertion*

*frameshift* causing immediate nonsense

Figure 17.22 The signal mechanism for targeting proteins to the ER

A polypeptide destined for endomembrane system or for secretion from cell begins with a signal peptide. Fig. shows synthesis of a secretory protein and its simultaneous import into the ER. In the ER and then the Golgi, the protein will be processed further. Finally, a transport vesicle will convey it to the plasma membrane for release from the cell.

Figure 17.17 Schematic model showing binding sites

A ribosome has an mRNA binding site and three tRNA binding sites, known as the A, P, and E sites.

Figure 17.14 Translation: the basic concept

As a molecule of mRNA is moved through a ribosome, codons are translated into amino acids, one by one. The interpreters are tRNA molecules, each type with a specific anticodon at one end and a corresponding amino acid at the other end. A tRNA adds its amino acid cargo to a growing polypeptide chain when the anticodon hydrogen-bonds to a complementary codon on the mRNA.

Figure 17.25 Coupled transcription and translation in bacteria

In bacterial cells, the translation of mRNA can begin as soon as leading (5') end of mRNA molecule peels away from DNA template. The micrograph (TEM) shows a strand of E. coli DNA being transcribed by RNA polymerase molecules. Attached to each RNA pol molecule is a growing strand of mRNA, which is already being translated by ribosomes. The newly synthesized polypeptides are not visible in the micrograph but are shown in diagram.

Figure 17.11 RNA processing: RNA splicing

The RNA molecule shown here codes for β-globin, one of the polypeptides of hemoglobin. The numbers under the RNA refer to codons; β-globin is 146 amino acids long. The β-globin gene and its pre-mRNA transcript have three exons, corresponding to sequences that will leave the nucleus as mRNA. During RNA processing, the introns are cut out and the exons spliced together. In many genes, the introns are much larger than the exons.

Figure 17.23 *Molecular basis of sickle-cell disease: a point mutation*

The allele that causes sickle-cell disease differs from the wild-type allele by a single DNA nucleotide pair. (1) In the DNA, the mutant (sickle-cell) template strand (top) has an A where the wild-type template has a T. (2) The mutant mRNA has a U instead of an A in one codon. (3) The mutant hemoglobin has a valine (Val) instead of glutamic acid (Glu).

Translation is a complex process in terms of its...

biochemistry and mechanics

In summary...

a gene can be defined as a region of DNA that can be expressed to produce a final functional product, either a polypeptide or an RNA molecule

*Point Mutations*

chemical changes in just one nucleotide pair of a gene

Spontaneous mutations can occur...

during DNA replication, recombination, or repair

Ribosomes are _________ and can ________.

identical; switch from free to bound

Anticodon

nucleotide triplet at one end of a tRNA molecule that base-pairs with a particular complementary codon on an mRNA molecule

A cell translates an mRNA message into protein with the help of...

tRNA

Genetic information flows from mRNA to protein through... START 17.4

the process of translation

The change of a single nucleotide in a DNA template strand can lead to...

the production of an abnormal protein

*Exon Shuffling*

this process may result in the evolution of new and potentially beneficial proteins

*In addition to binding site for mRNA, each ribosome has three binding sites for tRNA...*

• *P site*: holds tRNA carrying the growing polypeptide chain • *A site*: holds tRNA carrying the next amino acid to be added to the chain • *E site*: discharged tRNAs leave ribosome from here

*Transfer RNA (tRNA)*

• RNA molecule that functions as a translator between nucleic acid (DNA/RNA) and protein languages by carrying specific amino acids from cytoplasmic pool of amino acids to growing polypeptide in ribosome, where they recognize the appropriate codons in the mRNA • in eukaryotes, tRNA is made in nucleus and then travels from nucleus to cytoplasm where translation occurs • in both bacteria and eukaryotes, each tRNA is used repeatedly, picking up designated amino acid in cytosol, depositing this cargo onto polypeptide chain at ribosome and then leaving ribosome, ready to pick up another amino acid • consists of single RNA strand only about 80 nucleotides long

*Nucleotide-Pair Insertions and Deletions*

• addition or losses of nucleotide pairs in a gene • have disastrous effect on resulting protein more often than substitutions do • may alter the reading frame, producing a *frameshift mutation*

Polyribosomes

• also called polysome • group of several ribosomes attached to, and translating, the same single mRNA molecule simultaneously • enable a cell to make many copies of a polypeptide very quickly

*Elongation* (also see other set)

• amino acids are added one by one to previous amino acid at C-terminus of the growing chain • each addition involves participation of several proteins called *elongation factors* and occurs in a three-step cycle: codon recognition, peptide bond formation, translocation • energy expenditure occurs in 1st and 3rd steps • hydrolysis of GTP plays important role

*Bound Ribosomes*

• attached to cytosolic side of ER or to nuclear envelope • make proteins of endomembrane system and proteins secreted from the cell (ex. insulin)

Comparing Gene Expression in Bacteria, Archaea, and Eukarya START 17.6

• bacteria and eukarya differ in their RNA polymerases, termination of transcription, and ribosomes; archaea tend to resemble eukarya in these respects • bacteria can simultaneously transcribe and translate the same gene because they don't have a nucleus • in eukarya, transcription and translation are separated by the nuclear envelope • in archaea, transcription and translation are likely coupled

*Initiation* (also see other set)

• brings together mRNA, a tRNA bearing the first amino acid of the polypeptide, and the two subunits of a ribosome • union of mRNA, initiator tRNA and small ribosomal subunit is followed by attachment of large ribosomal subunit, completing the *translocation initiation complex* • proteins called *initiation factors* are required to bring all these components together • cell also expends energy obtained by hydrolysis of GTP molecule to form initiation complex

*Ribozymes*

• catalytic RNA molecules (not proteins) that function as enzymes and can splice RNA • discovery of ribozymes rendered obsolete the belief that all biological catalysts were proteins

*Nonsense Mutations*

• changes an amino acid codon to a stop codon, causing translation to be terminated prematurely • results in shorter and usually nonfunctional protein

Mutations START 17.5

• changes in the genetic material of a cell or virus • ultimate source of new genes...responsible for huge diversity of genes found among organisms

*Polypeptide synthesis always begins in the ____________, and finishes in the __________ unless _____________.*

• cytosol • cytosol • unless the polypeptide signal the ribosome to attach to the ER

Molecules of tRNA are not identical...

• each carries a specific amino acid on one end • each has an *anticodon* on the other end

*Termination*

• elongation continues until a stop codon in mRNA reaches the A site of the ribosome and the A site accepts a *release factor* • termination requires GTP hydrolysis and additional protein factors

Bacterial and eukaryotic ribosomes are somewhat similar but have significant differences...

• eukaryotes' are slightly larger and differ somewhat from bacterial ribosomes in their molecular composition • some antibiotic drugs specifically target bacterial ribosomes without harming eukaryotic ribosomes

Ribosomes

• facilitate specific coupling of tRNA anticodons with mRNA codons in protein synthesis • two ribosomal subunits (large and small) are made of proteins and *ribosomal RNA (rRNA)*

*Aminoacyl-tRNA Synthetases* (also see other set)

• family of related enzymes that carry out correct matching up of tRNA and amino acid • synthetase catalyzes covalent attachment of amino acid to its tRNA in a process driven by hydrolysis of ATP

Wobble

• flexible base pairing at third base of a codon • allows some tRNAs to bind to more than one codon

*Silent Mutation*

• have no effect on amino acid produced by a codon because of redundancy in the genetic code • change in nucleotide pair may transform one codon into another that is translated into the same amino acid • has no observable effect on phenotype

Spliceosome

• in some cases, RNA splicing is carried out by spliceosomes • consist of a variety of proteins and several snRNPs that recognize the splice sites

*RNA Processing* START 17.3

• modification of RNA primary transcripts, including splicing out of introns, joining together of exons and alteration of 5' and 3' ends • enzymes in eukaryotic nucleus modify pre-mRNA through this process before the genetic messages are dispatched to the cytoplasm

*5' Cap*

• modified form of a guanine nucleotide added onto 5' end after transcription of first 20-40 nucleotides • is not translated into protein

Ribosomal RNA (rRNA)

• molecules that, together with proteins, make up ribosomes • most abundant type of RNA

*Frameshift Mutation*

• mutation in which insertion or deletion of nucleotides alters reading frame of genetic message, the triplet grouping of nucleotides on the mRNA that is read during translation • will occur whenever the number of nucleotides inserted or deleted is not a multiple of three

*3 Nucleotide-Pair Deletion*

• no frameshift, but one amino acid missing • 3 nucleotide-pair insertion (not shown) would lead to an extra amino acid

Completing and Targeting the Functional Protein

• often translation is not sufficient to make a functional protein • polypeptide chains are modified after translation or targeted to specific sites in the cell

Exons

• other regions • eventually *ex*pressed, usually by being translated into amino acid sequences

*Signal-Recognition Particle (SRP)*

• protein-RNA complex that binds to and recognizes the signal peptide as it emerges from the ribosome • brings the signal peptide and its ribosome to a receptor protein built into the ER membrane

*Domain*

• proteins often have a modular architecture consisting of discrete structural and functional regions called domains • in many cases, different exons code for the different domains in a protein • *Figure 17.13*: Correspondence between exons and protein domains

*Poly-A Tail*

• sequence of 50-250 adenine nucleotides added onto 3' end of pre-mRNA molecule • is not translated into protein