Chapter 14 ARG From gene to protein

What are three properties of RNA that allow it to function as an enzyme?

1. Because RNA is single-stranded, a region of an RNA molecule may base-pair with a complementary region elsewhere in the same molecule, which gives the molecule a particular three-dimensional structure. A specific structure is essential to the catalytic function of ribozymes, just as it is for enzymatic proteins. 2. Like certain amino acids in an enzymatic protein, some of the bases in RNA contain functional groups that may participate in catalysis. 3. This ability of RNA to hydrogen-bond with other nucleic acid molecules (either RNA or DNA) adds specificity to its catalytic activity.

Describe at least three types of post-translational modifications.

1. Certain amino acids may be chemically modified by the attachment of sugars, lipids, phosphate groups, or other additions. 2. Enzymes may remove one or more amino acids from the leading (amino) end of the polypeptide chain. 3. Two or more polypeptides that are synthesized separately may come together, becoming the subunits of a protein that has quaternary structure.

Describe the action of different types of chemical mutagens.

1. Chemicals that are similar to normal DNA nucleotides but that pair incorrectly during DNA replication 2. Chemicals that interfere with correct DNA replication by inserting themselves into the DNA and distorting the double helix 3. Chemicals that cause chemical changes in bases that change their pairing properties

name the three stages of transcription and briefly describe each stage.

1. Initiation 2. Elongation 3. Termination

cite two significant findings resulted from the research of Beadle and Tatum

1. Metabolic defects are linked to defective genes 2. Support for the one gene-one enzyme hypothesis

Let's now take a closer look at initiation. Read the paragraph titled "RNA Polymerase Binding and Initiation of Transcription" carefully. List three important facts about the promoter here.

1. Promoter of a gene includes within it the transcription start point. 2. Promoter typically extends several dozen of more nucleotide pairs upstream from the start point. 3. RNA polymerase binds in a precise location and orientation on the promoter.

From the first paragraph in this section, find three ways in which RNA differs from DNA.

1. RNA contains ribose instead of deoxyribose as its sugar. 2. RNA has the nitrogenous base uracil rather than thymine. 3. An RNA molecule usually consists of a single strand rather than DNA's double strand

To the right of the figure, explain the three stages of initiation that are shown.

1. The tRNA carrying methionine attaches to the small ribosomal unit. 2. Together, they bind to the 5 end of the mRNA by recognizing the 5 GTP cap. 3. they, they "walk" along the mRNA in the 3 direction, stopping when they reach the start codon.

What are three important functions of the 5' cap and poly-A tail?

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

Describe two important ways in which bacterial and eukaryotic gene expression differ.

1. Transcription is terminated differently in bacteria and eukaryotes. 2. In the absence of a nucleus, bacterial cells can simultaneously transcribe and translate the same gene, and the newly made protein can quickly diffuse to its site of function. The eukaryotic cell's nuclear envelope segregates transcription from translation and provides a compartment for extensive RNA processing. This processing state includes additional steps whose regulation can help coordinate the eukaryotic cell's elaborate activities.

How many different aminoacyl-tRNA synthetases are there?

20

How many nucleotides are required to code for these 20 amino acids?

3

Here is a short DNA template. Below it, assemble the complementary mRNA strand.

3'A C G A C C A G T A A A 5' 5' U G C U G G U C A U U U 3'

How many codons are there above? Label one codon.

4

How many nucleotide bases are there? How many amino acids?

4 20

Of the 64 possible codons, how many code for amino acids?

61

So, the language of DNA is a triplet code. How many unique triplets exist?

64

How can a nucleotide-pair substitution result in a silent mutation?

A change in the nucleotide pair may transform one codon into another that is translated into the same amino acid. This mutation has no observable effect on the phenotype.

Define a mutation in terms of molecular genetics.

A change in the nucleotide sequence of an organism's DNA or in the DNA or RNA of a virus

What are frameshift mutations?

A frameshift mutation is a mutation occurring when nucleotides are inserted in or deleted from a gene and the number inserted or deleted is not a multiple of three, resulting in the improper grouping of the subsequent nucleotides into codons.

What is a gene? It used to be simply stated that one gene codes for one polypeptide. That definition has now been modified. Write below the broader molecular definition in use today

A gene is a region of DNA that can be expressed to produce a final functional product that is either a polypeptide or an RNA molecule.

What is the difference between a nonsense and missense mutation?

A nonsense mutation changes an amino acid codon to one of the three stop codons, resulting in a shorter and usually nonfunctional protein. A missense mutation is a nucleotide-pair substitution that results in a codon that codes for a different amino acid.

What is an anticodon?

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

What situation did Archibald Garrod suggest caused "inborn errors of metabolism"?

A person's inability to make a particular enzyme

Definepointmutations.

A point mutation is a change in a single nucleotide pair of a gene.

What is a polyribosome?

A polyribosome is a group of several ribosomes attached to, and translating, the same messenger RNA molecule.

What is a release factor? By what mechanism is termination accomplished?

A release factor is a protein shaped like an aminoacyl tRNA, which binds directly to the stop codon in the A site.Hydrolyzation is the mechanism by which termination is accomplished, releasing the polypeptide through the exit tunnel of the ribosome's large subunit.

Describe the structure of a eukaryotic ribosome.

A ribosome consists of a large subunit and a small subunit, each made up of proteins and one or more rRNAs.

Summarize the events of initiation. Include these components: small ribosomal subunit, large ribosomal subunit, mRNA, initiator codon, tRNA, Met, initiation complex, P site, and GTP. The figure below may help you.

A small ribosomal subunit binds to a molecule of mRNA. In a bacterial cell, the mRNA binding site on this subunit recognizes a specific nucleotide sequence on the mRNA just upstream of the start codon. An initiator tRNA, within the anticodon UAC, base-pairs with the start codon, AUG. This tRNA carries the amino acid methionine (Met). The arrival of a large ribosomal subunit completes the initiative complex. Proteins called initiation factors are required to bring all the translation components together. Hydrolysis of GTP provides the energy for the assembly. The initiator tRNA is in the P site; the A site is available to the tRNA bearing the next amino acid.

Transfer RNA has two attachment sites. What binds at each site? Sketch tRNA to indicate the two attachment sites, and note where complementary base pairing and hydrogen bonding occur to give it shape.

A specific anticodon binds at one end of tRNA, and a corresponding amino acid at the other end. See page 337 of your text for the labeled figure.

What is a transcription unit?

A transcription unit is a region of DNA that is transcribed into an RNA molecule.

What is the start codon?

AUG

Why is the genetic code said to be redundant but not ambiguous?

Although more than one codon may specify a particular amino acid, neither codon specifies any other amino acid.

What is a ribozyme?

An RNA molecule that functions as an enzyme, such as an intron that catalyzes its own removal during RNA splicing

What strategy did Beadle and Tatum adopt to test this hypothesis?

Beadle and Tatum bombarded the bread mold Neurospora with X-rays, and then looked among the survivors for mutants that differ in their nutritional needs from the wild-type bread mold.

What revision of detail (but not of basic principle) did this hypothesis undergo as more information was gained? Write this restatement and then box or highlight it. This is an important concept!

Because not all proteins are enzymes, and because each protein consists of two or more different polypeptide chains, each specified by its own gene, the hypothesis was revised. Beadle and Tatum's idea was restated as the one gene-one polypeptide hypothesis.

Write the central dogma of molecular genetics, as proclaimed by Francis Crick, in the box below.

DNA to RNA to protein

How does a prokaryotic ribosome differ from a eukaryotic ribosome? What is the medical significance of this difference?

Eukaryotic ribosomes are slightly larger in structure and differ somewhat from bacterial ribosomes in their molecular composition. Certain antibiotic drugs can inactivate bacterial ribosomes without inhibiting the ability of eukaryotic ribosomes to make proteins. These drugs, including tetracycline and streptomycin, are used to combat bacterial infections.

Describe one example Garrod used to illustrate his hypothesis.

Garrod gave as one example the hereditary condition called alkaptonuria. In this disorder, the urine is black because it contains the chemical alkapton, which darkens upon exposure to air. Garrod reasoned that most people have an enzyme that metabolizes alkapton, whereas people with alkaptonuria have inherited an inability to make that enzyme.

What is gene expression?

Gene expression is the process by which information encoded in DNA directs the synthesis of proteins or, in some cases, RNAs that are not translated into proteins and instead function as RNAs.

Distinguish between introns and exons. Perhaps it will help to remember this: Exons are expressed.

Introns are the noncoding segments of nucleic acid that lie between coding regions, also called intervening sequences Exons are the segments of nucleic acid that are eventually expressed by being translated into amino acid sequences.

What are some of the things that will result in a final-form functional protein?

Modifications of a protein after translation include protein folding, chemical modification of amino acids, enzymatic removal or rearrangement of amino acids, or the formation of quaternary- level proteins as in the case of hemoglobin.

Which organism did Beadle and Tatum use in their research? How did this organism's nutritional requirements facilitate this research?

Neurospora Wild-type Neurospora has modest food requirements. It can grow in the laboratory on a simple solution of inorganic salts, glucose, and the vitamin biotin, and incorporated into agar, a support medium. From this minimal medium, the mold cells use their metabolic pathways to produce all other molecules they need. Beadle and Tatum identified mutants that could not survive on minimal medium, apparently because they were unable to synthesize certain essential molecules from the minimal ingredients.

How were Neurospora spores treated to increase the mutation rate?

Neurospora spores were treated with X-rays to increase the mutation rate.

Describe Nirenberg's experiment in which he identified the first codon.

Nirenberg synthesized an artificial mRNA by linking identical RNA nucleotides containing uracil as their base.

Explain the concept of reading frame.

On an mRNA, the reading frame is the triplet grouping of ribonucleotides used by the translation machinery during polypeptide synthesis.

What are the two categories of mutagens?

Physical and chemical

In eukaryotes, what is the pre-mRNA called?

Primary transcript

Name the enzyme that uses the DNA template strand to transcribe a new mRNA strand.

RNA polymerase

Recall from Chapter 16 that DNA polymerase III adds new nucleotides to the template DNA strand to assemble each new strand of DNA. Both enzymes can assemble a new polynucleotide only in the 5'direction. Which enzyme, DNA polymerase III or RNA polymerase, does not require a primer to begin synthesis?

RNA polymerase

Now, summarize the events of elongation. Include these components: mRNA, A site, tRNA, codon, anticodon, ribozyme, P site, and E site. Again, the figure may help you.

See page 341 in your text for the labeled figure. The anticodon of an incoming aminoacyl tRNA base-pairs with the complementary mRNA codon in the A site. Hydrolysis of GTP increases the accuracy and efficiency of this step. An rRNA molecule of the large ribosomal subunit catalyzes the formation of a peptide bond between the amino group of the new amino acid in the A site and the carboxyl end of the growing polypeptide in the P site. This step removes the polypeptide from the tRNA in the P site and attaches it to the amino acid on the tRNA in the A site. The ribosome translocates the tRNA in the A site to the P site. At the same time, the empty tRNA in the P site is moved to the E site, where it is released. The mRNA moves along with its bound tRNAs, bringing the next codon to be translated into the A site.

Use the following figure to explain how proteins are targeted for the ER.

See page 343 in your text for the labeled figure. Polypeptide synthesis begins on a free ribosome in the cytosol. An SRP binds to a receptor protein in the ER membrane. This receptor is part of a protein complex that has a membrane pore and a signal-cleaving enzyme. The SRP leaves, and the polypeptide synthesis resumes, with simultaneous translocation across the membrane. The signal-cleaving enzyme cuts off the signal polypeptide. The rest of the completed polypeptide leaves the ribosome and folds into its final conformation.

You will be introduced to a number of small RNAs in this course. What type is the RNA in a snRNP?

Small nuclear RNA

What event is coded for by UAA, UAG, and UGA?

Stop

DNA is double-stranded, but for each protein, only one of these two strands is used to produce an mRNA transcript. What is the coding strand called?

Template strand

RNA processing occurs only in eukaryotic cells. The primary transcript is altered at both ends, and sections in the middle are removed. What happens at the 3' end?

The 3' end of the pre-mRNA molecule is also modified before the mRNA exits the nucleus. An enzyme adds 50-250 more adenine (A) nucleotides, forming a poly-A tail.

RNA processing occurs only in eukaryotic cells. The primary transcript is altered at both ends, and sections in the middle are removed. What happens at the 5' end?

The 5' end is synthesized first; it receives a 5' cap, a modified form of a guanine (G) nucleotide added onto the 5' end after transcription of the first 20-40 nucleotides.

What is the TATA box? How do you think it got this name?

The TATA box is a DNA sequence in eukaryotic promoters crucial in forming the transcription initiation complex. The name TATA box is from the thymine (T) and adenine (A) that make it up.

What comprises a transcription initiation complex?

The completed assembly of transcription factors and RNA polymerase bound to a promoter

What are the monomers of DNA and RNA? Of proteins?

The four types of nucleotides, which differ in their nitrogenous bases. amino acids

State the hypothesis formulated by George Beadle while studying eye color mutations in Drosophila.

The hypothesis stated that in Drosophila, each of the various mutations affecting eye color blocks pigment synthesis at a specific step by preventing production of the enzyme that catalyzes that step.

What commonly held idea was rendered obsolete by the discovery of ribozymes?

The idea that all biological catalysts are proteins

What is the consequence of alternative splicing of identical mRNA transcripts?

The number of different protein products an organism produces can be much greater than its number of genes.

SnRNPs band together in little snurp groups to form spliceosomes. How do spliceosomes work?

The spliceosome interacts with certain sites along an intron, releasing the intron, which is rapidly degraded, and joining together two exons that flanked the intron.

Complete the following table to summarize each process.

Transcription: Template: DNA Product synthesized: RNA Locatiion in eukaryotic cell: nucleus Translation: Template: mRNA Product synthesized: polypeptide Locatiion in eukaryotic cell: cytoplasm

What was the first codon-amino acid pair to be identified?

UUU (poly-U)-phenylalanine

Scientists expected to find one aminoacyl-tRNA synthetase per codon, but far fewer have been discovered. How does wobble explain this?

Wobble is flexibility in the base-pairing rules in which the nucleotide at the 5' end of a tRNA anticodon can form hydrogen bonds with more than one kind of base in the third position (3' end) of a codon. This flexibility explains why there are only about 45 tRNAs.

Much like transcription, we can divide translation into three stages. List them.

a. initiation b. elongation c. termination

explain the process of a specific amino acid being joined to a tRNA. be sure to use the words aminoacyl-tRNA synthetase, ATP, amino acid, and tRNA

active site of aminoacyl-TNRA synthetase binds the amino acid and ATP. ATP loses two P hgroups and joins amino acid to AMP. appropriate tNRA covalently bonds to an amino acid, displacing AMP. the tRNA charged with amino acid is released by the enzyme.

Three types of RNA are needed for protein synthesis. Complete the chart below.

mRNA description: Messenger RNA, Single-stranded. Function: Carries genetic material from the DNA to the protein-synthesizing machinery of the cell tRNA description: Transfer RNA, About 75 nucleotides long; folds into a clover-leaf shape. Function: Transfers amino acids from the cytoplasmic pool of amino acids to a growing polypeptide in a ribosome rRNA description: Ribosomal RNA Function: Together with proteins, makes up ribosomes; the most abundant type of RNA

What is always the first amino acid in the new polypeptide?

methionine

Study the figure and text carefully to explain how the splice sites are recognized.

snRNAs, part of the spliceosome complex, recognize specific nucleotide sequences on the intron and catalyze the process of intron removal. This is an excellent example of catalytic function in snRNA.

What are snRNPs? What two types of molecules make up a snRNP?

snRNPs are small nuclear ribonucleoproteins. snRNPs are made up of RNA and protein molecules

explain the functions of the A, P, and E sites on a ribosome

the A site holds the tRNA carrying the next amino acid to be added to the chain. the P site (start site) holds the tRNA carrying the growing polypeptide chain. the E site is where discharged tRNA's exit the ribosome.

Define each of these processes that are essential to the formation of a protein:

transcription: The synthesis of RNA using a DNA template translation: The synthesis of a polypeptide using the genetic information encoded in an mRNA molecule. There is a change of "language" from nucleotides to amino acids.