Unit 6 - Protein Synthesis

Question 1

Describe the structure of DNA

Answer 1

Purines: double ringed adenine and thymine

Pyrimidines: single ringed guanine and cytosine

5’ to 3’ order of nucleotide: phosphate group – 5-carbon sugar – nitrogenous base

The two strands of DNA are antiparallel.

10 base pairs for every DNA curl

Question 2

Describe the wobble effect. How does it help?

Answer 2

Pairing of the tRNA anticodon with the mRNA codon: once the first two positions are paired, the third base of the anticodon can typically pair with not only a complementary base: it “wobbles”. For example, the double-ringed G can pair with either a single-ringed U or C.

This allows mRNA to be translated with fewer than the 64 tRNAs that would be otherwise be required. Some wobble positions can pair with any of the four bases.

Question 3

Compare DNA and RNA

Answer 3

Question 4

Compare mRNA, tRNA, and rRNA

Answer 4

Messenger RNA:

• Intermediary between DNA and ribosome
• Translated into protein by ribosome
• RNA version of gene coded by DNA

Transfer RNA:

• Functions as the delivery system of amino acids to ribosomes
• At least one type of tRNA for each amino acid
• Shaped like a 3-leaf clover

Ribosomal RNA:

• Binds with proteins to form the ribosomes

Question 5

Before RNA leaves the nucleus, it is processed. What is an immature mRNA called? Explain this process

Answer 5

Immature mRNA: hnRNA (heterogenous nuclear)

A) 5’ Cap: the 5’ end is capped with a modified guanine nucleotide which serves as

1. An attach here signal from the small ribosomal subunit
2. Protection from enzymes

B) Poly-A Tail: 150 to 200 adenine nucleotides are added to the 3’ end which helps prevent degradation of the mRNA

C) Non-coding sequences (introns/intervening sequences) are removed. Exons/expressed sequences are kept

Question 6

Describe the possible function of introns

Answer 6

1. Plays a regulatory role in gene activity
2. Allows different types of cells in the same organism to different proteins from a common gene
3. Plays a role in the evolution of protein diversity

Question 7

Where is the lac operon found?

Answer 7

In the E. coli within your intestines. It converts lactose that you drink into energy.

Question 8

Descibe the layout of the lac operon

Answer 8

Left to right

Regulatory Gene: constantly transcribed and translated into an amino acid sequence that results in LacI repressor protein

Promotor: a DNA sequences that acts as a binding site for RNA polymerase

Operator: a binding site for LacI

Structural gene 1-3: these code for enzymes that break down lactose for energy

Question 9

Explain how the lac operon is negatively controlled

Answer 9

Lactose absent:

1. LacI binds to operator and blocks RNA polymerase from promoter
2. Genes cannot be transcribed, no waste enzymes produced

Lactose present:

1. Some lactose binds to LacI at control site, changing its shape and deactivating it
2. RNA polymerase accesses promoter and produces enzymes

Question 10

Explain how the lac operon is positively controlled

Answer 10

E. coli do not need lactose enzymes during high glucose concentrations because they utilize glucose instead.

Low glucose:

1. cAMP increases, CAP binds and increases structual gene transcription

High glucose:

1. cAMP decreases, CAP cannot bind and decreases structual gene transcription

Question 11

Where is the trp operon found?

Answer 11

Normally in E. coli in the intestine, but for E. coli that exists elsewhere it must manufacture its own tryptophan

Question 12

Explain how the trp operon is positively controlled

Answer 12

High tryptophan:

1. Tryptophanbind to trp repressor protein at control site, altering its shape and allowing it to bind to operator, stopping tryptophan production

Low tryptophan:

1. Trp repressor loses its shape without tryptophan binding, allowing RNA polymerase to bind to promoter and transcribe genes

Question 13

What is meant by saying tryptophan is a corerepressor?

Answer 13

Corerepressor: a molecule that binds to a repressor to activate it.

Question 14

Compare laq vs. trp operon

Answer 14

Lac operon is repressed during low lactose levels

Trp operon is repressed during high tryptophan levels

Question 15

Point mutations: (3)

Answer 15

Point Mutations: changes in one or two nucleotides in a single gene.

Includes substitution, insertion, and deletion

Question 16

What is the protein that breaks down mRNA when it is no longer needed?

Answer 16

Ribonuclease

Question 17

Substitution:

Answer 17

Substitution: a replacement of one nucleotide and its partner with another pair of nucleotides. Sometimes has no effect due to redundancy and the wobble effect

Question 18

Describe the two potential results from substitution

Answer 18

Missense mutation: the altered codon still codes for an amino acid and still makes sense but not always the intended sense

Nonsense mutation: the altered codon codes for premature termination (stop codon) creating a non-functional protein

Question 19

Insertions and deletions:

Answer 19

Insertions and Deletions: addition or loss of one or more nucleotide

Question 20

Duplication:

Answer 20

Duplication: a gene sequence in excess of its normal amount in a chromosome (ex., deletion from one chromosome is inserted in its homologue)

Question 21

Inversion:

Answer 21

Inversion: a chromosome segment that separated from the chromosome and was reinserted at the same place but inverse

Question 22

Translocation:

Answer 22

Translocation: the transfer of part of 1 chromosome (nonhomologous)

Question 23

What is it called when a ribosome walks down the mRNA?

Answer 23

Translocation

Question 24

Describe how chromosomes are packaged in the nucleus

Answer 24

1. Every 200 nucleotides, the DNA is coiled around a core group of stabilizing proteins called histones (+ive) which are attracted to -ive DNA.
   
   • This complex of histones enveloped by coiled DNA is called a nucleosome
2. Next, the series of nucleosomes coil into chromatin fibres
3. These fibres fold into the final chromatin structure via supercoiling
4. One DNA helix forms each chromosome

Question 25

How do you determine how many cuts with be made by a restriction enzyme in a set of DNA?

Answer 25

To tell how many cuts: 75000 (or simply the number of nucleotides in the DNA) / 4^(length of nucleotide to be cut).

Question 26

Explain transcriptional control

Answer 26

* Regulate which genes are transcribed * Control the rate at which transcription occurs.

Question 27

Explain posttranscriptional control

Answer 27

* Converting hnRNA to mRNA

Question 28

Explain translational control

Answer 28

* Controls how often and how rapidly mRNA transcripts will be translated into proteins. * Affects the length of time it takes for mRNA to be activated * Affects the speed at which enzymes destroy mRNA.

Question 29

Explain posttranslational control

Answer 29

* Before many proteins become functional, they must pass through the cell membrane. Several control mechanisms affect this rate.

Question 30

How many hydrogen bonds do the nitrogenous bases have?

Answer 30

Adenine and thymine have 2, guanine and cytosine have 3. So, adenine and thymine are easier to separate.

Question 31

Define biotechnology

Answer 31

**Biotechnology**: the use of biological substances to develop an agricultural, medicinal, environmental, etc. product or process (synthetic hormones, insulin, food)

Question 32

What is recombinant DNA? Provide natural examples.

Answer 32

Recombinant DNA refers to new genetic material constructed from DNA of different organisms Natural examples (evolution): * Combinations of genes between different organisms (fertilization/crossing over) * Bacteria: during transformation, they picked up free DNA * Virus: sometimes transfers genes into eukaryotic cell host

Question 33

State 3 tools used to create recombinant DNA in a lab. What are methylases?

Answer 33

**Plasmids**: ring of DNA found in bacteria that carries accessory genes which replicate independently of the useful DNA **DNA Ligase**: an enzyme used to "glue" fragments by condensation that have been cut by the same restriction enzyme **Restriction Enzyme**: enzymes that protect bacteria from bacteriophage DNA injections, make palindromic cuts. * *Methylases*: enzymes that modify recognition sites by placing a methyl group on a base. This prevents the cutting of that portion of the DNA; prevents digestion of bacteria’s own DNA

Question 34

Explain how plasmids, ligase, and restriction enzymes are used together

Answer 34

1. Combining these three devices allows for the _duplication of human DNA_ 2. Plasmid and the human DNA sample are treated with the same restriction enzyme. 3. The enzyme cuts out the specific sequence of the human DNA which is then inserted into the plasmid, replacing the plasmid DNA that was snipped as well. 4. Ligase seals the ends. The bacteria are allowed to replicate.

Question 35

What is gel electrophoresis? Describe the steps it requires

Answer 35

Gel electrophoresis is used to separate charged molecules on the bases of size through a gel meshwork. DNA is negatively charged due to its phosphate groups. Steps 1. DNA is cleaved by restriction enzymes into fragments of different lengths 2. A gel is used which is comprised of a buffer and agarose 3. A current causes DNA to move from the negative to positive terminal. Smaller fragments travel faster

Question 36

What is RFLP

Answer 36

**Restriction Fragment Length Polymorphism**: any difference in DNA sequence that can be detected between individuals

Question 37

Explain southern blotting. What is the x-ray used called?

Answer 37

A procedure that allows DNA in a gel to be transferred to a nylon membrane. A membrane is placed on the gel and the DNA is blotted onto it (positive electrode behind membrane). The DNA is then stained. The resulting picture generated via x-ray is called an **autoradiogram**.

Question 38

Explain the process of PCR

Answer 38

Polymerase chain reactionis used to make many copies of a specific DNA region in *vitro.* 1. PCR relies on a DNA polymerase, Taq polymerase 2. The experimenter determines the region of DNA that will be amplified by the primers she or he chooses. 3. The DNA primers must be complementary to the target area. 4. When the primers are bound to the template, they are extended by Taq polymerase. 5. The new DNA that’s made in one round can serve as a template in the next round of DNA synthesis.

Question 39

Describe chain termination. What is it also known as?

Answer 39

A.k.a. DNA Sequencing or Sanger Dideoxy Method 1. Treat double stranded DNA to become single stranded 2. Add primer to end of DNA 3. Add 4 identical copies of DNA to 4 tubes with each containing DNA polymerase, free nucleotides, and 1 **dideoxy nucleotide** (a nucleotide – A’, G’, C’, T’ – missing an OH on the 3’ carbon deoxyribose sugar, stopping DNA synthesis) 4. This creates many different fragments of DNA which are organized via gel electrophoresis and are read off from _bottom to top_ to get the complementary strand which is *again read off to get the original strand*.