Quiz

• Nucleotide?

• Added to which end?

• Lagging strand?

DNA pol III synthesizes leading strand continuously

Parental DNA DNA pol III starts DNA

synthesis at 3 end of primer, continues in 5 3 direction

Lagging strand synthesized in short Okazaki fragments, later joined by DNA ligase

Primase synthesizes a short RNA primer

53

5

5

5

3

3

Chapter 17Chapter 17

From Gene to Protein

Big Questions

• How does your body produce insulin?

• How does your offspring know how to make insulin?

• Central “Dogma”

DNA > RNA > “Protein”

(Info > Message > Product)

Chapter 17 Study Guide Questions

The Connection between Genes and Proteins

1. Explain how RNA differs from DNA.

2. Briefly explain how information flows from gene to protein. Is the central dogma ever violated?

3. Distinguish between transcription and translation.

4. Compare where transcription and translation occur in bacteria and in eukaryotes.

5. Define “codon” and explain the relationship between the linear sequence of codons on mRNA and the linear sequence of amino acids in a polypeptide.

6. Explain what it means to say that the genetic code is redundant and unambiguous.

7. Explain the significance of the reading frame during translation.

Chapter 17 Study Guide Questions

The Synthesis and Processing of RNA

8. Explain how RNA polymerase recognizes where transcription should begin. Describe the role of the promoter, the terminator, and the transcription unit.

9. Explain the general process of transcription, including the three major steps of initiation, elongation, and termination.

10. Explain how RNA is modified after transcription in eukaryotic cells.

11. Define and explain the role of ribozymes. What three properties allow some RNA molecules to function as ribozymes?

12. Describe the functional and evolutionary significance of introns.

13. Explain why, due to alternative RNA splicing, the number of different protein products an organism can produce is much greater than its number of genes.

Chapter 17 Study Guide Questions

The Synthesis of Protein

14. Describe the structure and function of tRNA.

15. Explain how tRNA is joined to the appropriate amino acid.

16. Describe the structure and functions of ribosomes.

17. Describe the process of translation (including initiation, elongation, and termination) and explain which enzymes, protein factors, and energy sources are needed for each stage.

18. Describe the significance of polyribosomes.

19. Explain what determines the primary structure of a protein and describe how a polypeptide must be modified before it becomes fully functional.

20. Define “point mutations”. Distinguish between base-pair substitutions and base-pair insertions. Give an example of each and note the significance of such changes.

Today’s Tip: Ricin will kill you

Georgi Ivanov Markov

Inhibits ribosome translation of mRNA

1. Explain how RNA differs from DNA.

DNA

• Double-stranded

• ATCG

• Deoxyribose

RNA

• Single-stranded

• AUCG

• Ribose

2. Briefly explain how information flows from gene to protein. Is the central dogma ever violated?

Genes

Specific sequences of nucleotides

Proteins

Instructions carried in genes

Gene expression•DNA directs protein synthesis

Includes two stages: •transcription and translation

Cellular chain of command:

DNA RNA protein

3. Distinguish between transcription and translation.

Transcription• synthesis of RNA under the direction of DNA

Messenger RNA (mRNA)

Product of transcription

Translation• synthesis of a polypeptide• occurs under the direction of mRNA•_________are the sites of translation

4. Compare where transcription and translation occur in bacteria and in eukaryotes.

Prokaryotes

Transcription – cytoplasm

Translation – cytoplasm

Eukaryotes

Transcription – Nucleus

Translation – cytoplasm

LE 17-3-1LE 17-3-1

TRANSCRIPTIONDNA

Prokaryotic cell

LE 17-3-2LE 17-3-2

TRANSCRIPTIONDNA

Prokaryotic cellRibosome

Polypeptide

mRNA

Prokaryotic cell

LE 17-3-3LE 17-3-3

TRANSCRIPTION

TRANSLATION

DNA

mRNA

Ribosome

Polypeptide

DNA

Prokaryotic cell

Nuclearenvelope

TRANSCRIPTION

Eukaryotic cell

LE 17-3-4LE 17-3-4

TRANSCRIPTION

TRANSLATION

DNA

mRNA

Ribosome

Polypeptide

DNA

Pre-mRNA

Prokaryotic cell

Nuclearenvelope

mRNA

TRANSCRIPTION

RNA PROCESSING

Eukaryotic cell

LE 17-3-5LE 17-3-5

TRANSCRIPTION

TRANSLATION

DNA

mRNA

Ribosome

Polypeptide

DNA

Pre-mRNA

Prokaryotic cell

Nuclearenvelope

mRNA

TRANSLATION

TRANSCRIPTION

RNA PROCESSING

Ribosome

Polypeptide

Eukaryotic cell

Codons

• mRNA base triplets

• specifies specific amino acid

• amino acid placed at the corresponding position along a polypeptide

Template strand

• provides a template for ordering the sequence of nucleotides in an RNA transcript

5. Define “codon” and explain the relationship between the linear sequence of codons on mRNA and the linear sequence of amino acids in a polypeptide.

5. Define “codon” and explain the relationship between the linear sequence of codons on mRNA and the linear sequence of amino acids in a polypeptide.

Triplet code

• nonoverlapping

• three-nucleotide words

• code for amino acids

Example:

• AGT on DNA strand

• results in the placement of the amino acid serine

• at the corresponding position of the polypeptide to be produced

6. Explain what it means to say that the genetic code is redundant and unambiguous.

How many amino acids?

20

How many codons?

64

What does that imply?

6. Explain what it means to say that the genetic code is redundant and unambiguous.

Redundant but not ambiguous (?)

1. One amino acid can have more than one codon

e.g., UCU, UCC, UCA, etc., all code for Serine

2. No codon specifies more than one amino acid

7. Explain the significance of the reading frame during translation.

The fat cat ate the ratReading Frame

• Codons must be read in the correct reading frame

• What happens if you are off by one letter?

• Frame Shift Error

Practice

What amino acid does CAC code for?

• AGA?

• CGU?

• Three code for stop

• AUG codes for begin

Evolution of the Genetic Code

The genetic code is nearly universal

What does that mean?

• shared by the simplest bacteria to the most complex animals

What does that imply?

Luciferase

9. Explain the general process of transcription, including the three major steps of initiation, elongation, and termination.

The three stages of transcription:

1) Initiation

2) Elongation

3) Termination

Molecular Components of Transcription

RNA Polymerase

pries the DNA strands apart and hooks together the RNA nucleotides

• RNA synthesis follows the same base-pairing rules as DNA

• except __?__ substitutes for thymine

8. Explain how RNA polymerase recognizes where transcription should begin. Describe the role of the promoter, the terminator, and the transcription unit.Initiation

Promoter• DNA sequence where RNA polymerase attaches

Transcription Unit• stretch of DNA that is transcribed

Terminator• DNA sequence found only on prokaryotes

Animation: Transcription

LE 17-7LE 17-7Promoter

5¢3¢

Transcription unit

3¢5DNA

Start pointRNA polymerase

Initiation

3¢5

5¢3¢

UnwoundDNA

RNAtran-script

Template strandof DNA

Elongation

RewoundDNA

3¢5

5¢3¢ 3¢

5¢

RNAtranscript

LE 17-7LE 17-7Promoter

3¢5

Transcription unit

DNA

InitiationRNA polymerase

Start point

Template strandof DNA

RNAtran-script

UnwoundDNA

Elongation

3¢

3¢

5¢3

5¢

5¢

3¢ 5¢

RewoundDNA

5¢ 3¢

3¢5¢ 3¢5¢

RNAtranscript Termination

3¢5¢

5¢ 3¢Completed RNA transcript

Elongation of the RNA Strand

RNA polymerase• untwists the double helix• 10 to 20 bases at a time• 40 nucleotides per second (eukaryotes)• can be transcribed simultaneously by several RNA polymerases

Why?

Termination of Transcription

Mechanisms of termination are different in prokaryotes and eukaryotes

Prokaryotes

• polymerase stops transcription at the end of the terminator sequence

Eukaryotes

• polymerase continues transcription after the pre-mRNA is cleaved

• Polymerase eventually falls off the DNA

10. Explain how RNA is modified after transcription in eukaryotic cells.• Does the pre-mRNA now leave the nucleus?

• How is the pre-mRNA modified?

Alteration of mRNA EndsEach end of a pre-mRNA molecule is modified in a particular way:

– The 5 end receives a modified nucleotide cap

– The 3 end gets a poly-A tail

These modifications share several functions:

– They seem to facilitate the export of mRNA

– They protect mRNA from hydrolytic enzymes

– They help ribosomes attach to the 5’ end

Split Genes and RNA Splicing

Intervening sequences (Introns)

• long noncoding stretches - removed

Exons

• shorter coding sections – will be expressed

RNA splicing

• removes introns and joins exons

• creating an mRNA molecule with a continuous coding sequence

RNA duplicating RNA, a step closer to the origin of life By Yun Xie |http://arstechnica.com/science/news/2011/04/investigations-into-the-ancient-rna-world.ars

NASA JPL According to the “RNA world” model of life's origin, RNA performed all of the operations that are essential to life. RNA alone passed on genetic information and catalyzed the reactions of basic metabolism; DNA and proteins were not in the picture. The RNA world hypothesis is an appealingly simple model for simple early life forms, since it allows the complex array of biochemical interactions among proteins, DNA, and RNA to evolve gradually.

Our current natural world no longer uses RNA enzymes that act on their own to perform most biological functions. To better understand ancient RNA enzymes, modern scientists have to rely on proxies, like engineered RNA "ribozymes" that have catalytic functions without the need for proteins. However, scientists have had trouble creating a proxy for the first self-replicating molecule, or even an RNA ribozyme that can copy an RNA that's long enough to have further biological functions. Aniela Wochner and her coauthors have overcome that difficulty. In a recent issue of Science, they report the creation of an RNA ribozyme that synthesizes complex RNAs, including RNAs that act as ribozymes and perform a biological function. Previously, the leading RNA polymerase ribozyme, called R18, could only transcribe RNAs up to 14 bases long (as a frame of reference, R18 itself is about 196 bases long). It was also highly template-dependent, meaning it could only copy certain sequences of RNA. To establish early life on Earth, a ribozyme would need to be able to make a variety of RNA sequences of adequate length, including something long enough to synthesize itself. Wochner and her colleagues sought to engineer a superior RNA ribozyme by modifying R18. First, they wanted to improve the interactions among the template RNA, the ribozyme, and the primer sequence that starts the copying. To make RNA, the ribozyme has to recognize the primer and the template, which base-pair with one another. Then, the ribozyme catalyzes the addition of new bases onto the primer, making an RNA sequence that is complementary to the template. Scientists have proposed that the one end of the R18 ribozyme interacts with the primer and template. So, Wochner and her colleagues appended a random sequence into the 5’-end of R18 and selected for improved RNA polymerase activity.They found one ribozyme (named C19) that did better than R18 on a specific, short template, but it didn't work well on longer templates. They further modified C19 by making truncated variants of its sequence and screened for improved activity on longer templates. They found one variant (the ribozyme tC19) that can extend primers by up to 95 bases with favorable templates.The final obstacle was the fact that it only worked well on favorable template sequences—the researchers wanted a ribozyme that will be able to copy a diverse range of RNA templates, not just a few favorable ones. To find one, they made 50 million randomly mutated R18 sequences, did numerous rounds of selections, and found a combination of mutations that improved the recognition of diverse templates. They applied those mutations to the tC19 ribozyme, creating the RNA polymerase ribozyme tC19Z (198-bases long).Ribozyme tC19Z synthesizes longer RNA sequences and can work with a greater range of primer-template combinations than any of the previous ribozymes. Wochner and her colleagues were able to use tC19Z to synthesize a minimal version of the hammerhead ribozyme (an RNA that binds to and cleaves an RNA substrate). The synthesized hammerhead ribozyme had catalytic activity, as it was able to cleave an RNA substrate at the expected location in the sequence.Wochner and her coauthors have significantly expanded our abilities to engineer RNA polymerase ribozymes; however, further improvements are still necessary. For example, tC19Z probably cannot synthesize something of its own size in a reasonable amount of time. Nevertheless, it's impressive that the researchers were able to select for such drastic improvements on R18, as the sequence hasn't seen a significant upgrade since its creation almost a decade ago. Their work lead us closer to understanding ribozymes that could have existed in early Earth.Science, 2011. DOI: 10.1126/science.1200752 (About DOIs)

Concept 17.4: Translation is the RNA-directed synthesis of a polypeptide: a closer look

Transfer RNA (tRNA)

•translates an mRNA message into protein

•Molecules of tRNA are not identical:

– Each carries a specific amino acid on one end

– Each has an anticodon on the other end

14. Describe the structure and function of tRNA.

tRNA molecule

• single RNA strand

• ~ 80 nucleotides long

• cloverleaf shape

• twists and folds into a three-dimensional molecule

How?

• hydrogen bonds

ACC

“problems”

A. How do you make sure that the correct amino acid to connects to the correct tRNA?

B. How do you make sure that the correct tRNA matches up with the correct codon?

15. Explain how tRNA is joined to the appropriate amino acid.

Accurate translation requires two steps:

First step:

– correct match between a tRNA and an amino acid

– done by the enzyme aminoacyl-tRNA synthetase (20 types)

Second step:

- correct match between the tRNA anticodon and an mRNA codon

16. Describe the structure and functions of ribosomes.

Ribosomes

facilitate specific coupling of tRNA anticodons with mRNA codons in protein synthesis

Two ribosomal subunits (large and small)

made of proteins and ribosomal RNA (rRNA)

You have brucellosis. What do you do?

• Tetracycline:

• works by binding specifically to the 30S ribosome of the bacteria

• preventing attachment of the aminoacyl tRNA to the RNA-ribosome complex

•A ribosome has three binding sites for tRNA:

– The P site

– holds the growing polypeptide chain

– The A site

– holds the next amino acid to be added

– The E site

– is the exit site, where discharged tRNAs leave the ribosome

17. Describe the process of translation (including initiation, elongation, and termination) and explain which enzymes, protein factors, and energy sources are needed for each stage.

•The three stages of translation:

– Initiation

– Elongation

– Termination

Ribosome Association and Initiation of Translation

Initiation stage

• brings together mRNA, a tRNA with the first amino acid, and the two ribosomal subunits

Ribosome Association and Initiation of Translation

•First, a small ribosomal subunit binds with mRNA and a special initiator tRNA

Ribosome Association and Initiation of Translation

Then the small subunit moves along the mRNA until it reaches the start codon (AUG)

Ribosome Association and Initiation of Translation

Proteins called initiation factors bring in the large subunit so the initiator tRNA occupies the P site

Elongation of the Polypeptide Chain

Elongation

• Amino acids added one by one

• Each addition involves proteins called elongation factors

•Occurs in three steps:

1. codon recognition

2. peptide bond formation

3. translocation

LE 17-18LE 17-18

Ribosome ready fornext aminoacyl tRNA

mRNA

5¢

Amino endof polypeptide

E

Psite

Asite

3¢

2

2 GDP

E

P A

GTP

GTP

GDP

E

P A

E

P A

three steps:1- codon recognition2 -peptide bond formation3- translocation

LE 17-19LE 17-19

3¢

The release factor hydrolyzes thebond between the tRNA in theP site and the last amino acid of thepolypeptide chain. The polypeptideis thus freed from the ribosome.

The two ribosomal subunitsand the other componentsof the assembly dissociate.

Releasefactor

Stop codon(UAG, UAA, or UGA)

5¢

3¢

5¢

3¢

5¢

Freepolypeptide

When a ribosome reaches a stopcodon on mRNA, the A site of theribosome accepts a protein calleda release factor instead of tRNA.

Termination of Translation

Ribosome acts as one large Ribozyme

18. Describe the significance of polyribosomes.

Polyribosomes

– make many copies of a polypeptide very quickly

19. Explain what determines the primary structure of a protein and describe how a polypeptide must be modified before it becomes fully functional.

Primary structure

Amino acid sequence

Polypeptide chains are modified after translation

Post-translational modifications

–Removal of amino acids• AUG

–Splitting of polypeptide chain

• insulin

Targeting Polypeptides to Specific Locations

Two populations of ribosomes are evident in cells:

1. free ribsomes (in the cytosol)

2. bound ribosomes (attached to the ER)

Free ribosomes mostly synthesize proteins that function in the cytosol

Targeting Polypeptides to Specific Locations

Bound ribosomes:• endomembrane system• secreted from the cell

Ribosomes are identical and can switch from free to bound

Concept 17.5: RNA plays multiple roles in the cell: a review

Type of RNA Functions

Messenger RNA (mRNA)

Carries information specifying amino acid sequences of proteins from DNA to ribosomes

Transfer RNA (tRNA)

Serves as adapter molecule in protein synthesis; translates mRNA codons into amino acids

Ribosomal RNA (rRNA)

Plays catalytic (ribozyme) roles and structural roles in ribosomes

Type of RNA Functions

Primary transcript

Serves as a precursor to mRNA, rRNA, or tRNA, before being processed by splicing or cleavage

Concept 17.6: Comparing gene expression in prokaryotes and eukaryotes reveals key differences

Prokaryotic cells:• lack a nuclear envelope• allows translation to begin while transcription progresses

Eukaryotic cell:– The nuclear envelope

separates transcription from translation

– Extensive RNA processing occurs in the nucleus

LE 17-22LE 17-22

RNA polymerase

DNA

Polyribosome

RNApolymerase

Direction oftranscription

mRNA

0.25 mm

DNA

Polyribosome

Polypeptide(amino end)

Ribosome

mRNA (5¢ end)

Coupled transcription and translation in bacteria

20. Define “point mutations”. Distinguish between base-pair substitutions and base-pair insertions. Give an example of each and note the significance of such changes.

Mutations

• changes in the genetic material of a cell or virus

Point mutations

• chemical changes in just one base pair of a gene

The change of a single nucleotide in a DNA template strand leads to production of an abnormal protein

LE 17-23LE 17-23

Wild-type hemoglobin DNA

mRNA

3¢ 5¢ 5¢3¢

5¢ 3¢ 3¢5¢

Mutant hemoglobin DNA

mRNA

Normal hemoglobin Sickle-cell hemoglobin

Substitutions

Base-pair substitution• replaces one nucleotide and its partner with another pair of nucleotides• can cause missense or nonsense mutations

Missense• mutations still code for an amino acid, but not necessarily the right amino acid

Nonsense mutations• change an amino acid codon into a stop codon, nearly always leading to a nonfunctional protein

Missense mutations are more common

Insertions and Deletions

Insertions and deletions

• additions or losses of nucleotide pairs in a gene

• have a disastrous effect on the resulting protein more often than substitutions do

• may alter the reading frame, producing a frameshift mutation

Mutagens

Spontaneous mutations • may occur during DNA replication, recombination, or repair

Mutagens • physical or chemical agents that can cause mutations

– Mutagenic radiation, ultraviolet light (physical)

– Cancer-causing chemicals