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Quiz. Nucleotide? Added to which end? Lagging strand?. DNA pol III synthesizes leading strand continuously. 3 . 5 . Parental DNA. DNA pol III starts DNA synthesis at 3  end of primer, continues in 5   3  direction. 5 . 3 . 5 .

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slide1
Quiz
  • Nucleotide?
  • Added to which end?
  • Lagging strand?
slide2

DNA pol III synthesizes leading strand continuously

3

5

Parental DNA

DNA pol III starts DNA synthesis at 3 end of primer, continues in 5 3 direction

5

3

5

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

Primase synthesizes a short RNA primer

3

5

chapter 17

Chapter 17

From Gene to Protein

big questions
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
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 questions1
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 questions2
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
Today’s Tip: Ricin will kill you

Georgi Ivanov Markov

Inhibits ribosome translation of mRNA

1 explain how rna differs from dna
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
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
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
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 1

LE 17-3-1

DNA

TRANSCRIPTION

Prokaryotic cell

le 17 3 2

LE 17-3-2

DNA

TRANSCRIPTION

mRNA

Ribosome

Prokaryotic cell

Polypeptide

Prokaryotic cell

le 17 3 3

LE 17-3-3

DNA

TRANSCRIPTION

mRNA

Ribosome

TRANSLATION

Polypeptide

Prokaryotic cell

Nuclear

envelope

DNA

TRANSCRIPTION

Eukaryotic cell

le 17 3 4

LE 17-3-4

DNA

TRANSCRIPTION

mRNA

Ribosome

TRANSLATION

Polypeptide

Prokaryotic cell

Nuclear

envelope

DNA

TRANSCRIPTION

Pre-mRNA

RNA PROCESSING

mRNA

Eukaryotic cell

le 17 3 5

LE 17-3-5

DNA

TRANSCRIPTION

mRNA

Ribosome

TRANSLATION

Polypeptide

Prokaryotic cell

Nuclear

envelope

DNA

TRANSCRIPTION

Pre-mRNA

RNA PROCESSING

mRNA

Ribosome

TRANSLATION

Polypeptide

Eukaryotic cell

slide18

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.

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
slide19

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
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 unambiguous1
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
7. Explain the significance of the reading frame during translation.

The fat cat ate the rat

Reading Frame

  • Codons must be read in the correct reading frame
  • What happens if you are off by one letter?
  • Frame Shift Error
practice
Practice

What amino acid does CAC code for?

  • AGA?
  • CGU?
  • Three code for stop
  • AUG codes for begin
evolution of the genetic code
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

slide25
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
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
slide27

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 7

LE 17-7

Promoter

Transcription unit

5

DNA

Start point

RNA polymerase

Initiation

5

Template strand

of DNA

RNA

tran-

script

Unwound

DNA

Elongation

Rewound

DNA

5

RNA

transcript

le 17 71

LE 17-7

Promoter

Transcription unit

5

3

DNA

Start point

RNA polymerase

Initiation

Template strand

of DNA

RNA

tran-

script

Unwound

DNA

Elongation

Rewound

DNA

RNA

transcript

Termination

Completed RNA transcript

elongation of the rna strand
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
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
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 ends
Alteration of mRNA Ends

Each 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 ribosomesattach to the 5’ end
split genes and rna splicing
Split Genes and RNA Splicing

Intervening sequences (Introns)

  • long noncoding stretches - removed

Exons

  • shorter coding sections – will be expressed

RNA splicing

  • removesintrons and joins exons
  • creating an mRNA molecule with a continuous coding sequence
slide35

RNA duplicating RNA, a step closer to the origin of lifeBy YunXie |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

Concept 17.4: Translation is the RNA-directed synthesis of a polypeptide: a closer look
14 describe the structure and function of trna
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

A

C

C

problems
“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
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-tRNAsynthetase (20 types)

Second step:

- correct match between the tRNAanticodon and an mRNA codon

16 describe the structure and functions of ribosomes
16. Describe the structure and functions of ribosomes.

Ribosomes

facilitate specific coupling of tRNAanticodons 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
You have brucellosis. What do you do?
  • Tetracycline:
  • works by binding specifically to the 30S ribosome of the bacteria
  • preventing attachment of the aminoacyltRNA to the RNA-ribosome complex
slide42
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
slide43
The three stages of translation:

Initiation

Elongation

Termination

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.

ribosome association and initiation of translation
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 translation1
Ribosome Association and Initiation of Translation
  • First, a small ribosomal subunit binds with mRNA and a special initiator tRNA
ribosome association and initiation of translation2
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 translation3
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 of the Polypeptide Chain

Elongation

  • Amino acids added one by one
  • Each addition involves proteins called elongation factors
  • Occurs in three steps:
  • codon recognition
  • peptide bond formation
  • translocation
le 17 18

LE 17-18

Amino end

of polypeptide

E

mRNA

P

site

A

site

Ribosome ready for

next aminoacyl tRNA

GTP

2

2 GDP

E

E

P

A

P

A

GDP

GTP

three steps:

1- codon recognition

2 -peptide bond formation

3- translocation

E

P

A

le 17 19

Release

factor

Free

polypeptide

Stop codon

(UAG, UAA, or UGA)

When a ribosome reaches a stop

codon on mRNA, the A site of the

ribosome accepts a protein called

a release factor instead of tRNA.

LE 17-19

Termination of Translation

The release factor hydrolyzes the

bond between the tRNA in the

P site and the last amino acid of the

polypeptide chain. The polypeptide

is thus freed from the ribosome.

The two ribosomal subunits

and the other components

of the assembly dissociate.

Ribosome acts as one large Ribozyme

18 describe the significance of polyribosomes
18. Describe the significance of polyribosomes.

Polyribosomes

  • make many copies of a polypeptide very quickly
slide52

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
Targeting Polypeptides to Specific Locations

Two populations of ribosomes are evident in cells:

  • free ribsomes (in the cytosol)
  • bound ribosomes (attached to the ER)

Free ribosomes mostly synthesize proteins that function in the cytosol

targeting polypeptides to specific locations1
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 6 comparing gene expression in prokaryotes and eukaryotes reveals key differences
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 22

LE 17-22

RNA polymerase

DNA

mRNA

Polyribosome

Direction of

transcription

0.25 mm

RNA

polymerase

DNA

Polyribosome

Polypeptide

(amino end)

Ribosome

mRNA (5¢ end)

Coupled transcription and translation in bacteria

slide59

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 23

LE 17-23

Wild-type hemoglobin DNA

Mutant hemoglobin DNA

mRNA

mRNA

Normal hemoglobin

Sickle-cell hemoglobin

substitutions
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

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
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
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