topic 2.7/7.1/7.2/7.3- DNA replication, transcription and translation

Question 1

describe the meselson and stahl experiment

Answer 1

DNA molecules were prepared using the heavier 15N and then induced to replicate in the presence of the lighter 14N

DNA samples were then separated via centrifugation to determine the composition of DNA in the replicated molecules

The results after two divisions supported the semi-conservative model of DNA replication

After one division, DNA molecules were found to contain a mix of 15N and 14N, disproving the conservative model
After two divisions, some molecules of DNA were found to consist solely of 14N, disproving the dispersive model

Question 2

primary structure of proteins

Answer 2

the sequence and number of amino acids in the polypeptide

Question 3

how many commonly occurring amino acids are there

Answer 3

20

Question 4

secondary structure of a protein

Answer 4

the formation of alpha helices and beta pleated sheets stabilised by hydrogen bonding

Question 5

what do hydrogen bonds form between in secondary structures of protein?

Answer 5

the carbonyl (C=O) group of one residue and the amino group (N-H) of an amino acid in another part of the chain

Question 6

tertiary structure of a protein

Answer 6

the further folding of the polypeptide stabilised by interactions between R groups

Question 7

state the 4 types of interaction between R groups (tertiary protein structure)

Answer 7

• +vely charged R-groups will interact with -vely charged R-groups
• hydrophobic AAs orientate themselves toward the centre of the polypeptide to avoid water contact, but hydrophilic AAs orientate themselves outward
• polar R-groups form H bonds with other polar R-groups
• R-group of AA cysteine forms a covalent bond with the R-group of another cysteine forming a disulphide bridge

Question 8

quaternary structure of a protein

Answer 8

exists in proteins with more than one polypeptide chain (and sometimes non-polypeptide components)

Question 9

define transcription

Answer 9

the synthesis of mRNA copied from the DNA base sequences by RNA polymerase

Question 10

name the 3 stages of translation

Answer 10

initiation, elongation, termination

Question 11

where does transcription begin?

Answer 11

at the promoter (a site in the DNA)

Question 12

what direction does transcription occur in?

Answer 12

the 3’ to 5’ direction

Question 13

define a cistron

Answer 13

the bit of the DNA that unwinds, so the region that codes for one allele

Question 14

describe the 5 steps of transcription

Answer 14

1. RNA polymerase binds to the promoter
2. it moves along the gene separating the DNA strand into single strands and pairing up mRNA nucleotides with complementary bases on the template/ antisense strand (in the 3’ to 5’ direction)
3. it forms covalent bonds between the RNA nucleotides
4. transcription stops at the end of the gene and the mRNA strand leaves through the nuclear pore

Question 15

in what direction does transcription occur

Answer 15

3’ to 5’ direction, following the antisense strand

Question 16

why do we call the genetic code degenerate? and universal?

Answer 16

there are more codons than amino acids and therefore some amino acids have more than one codon; it is the same across all kingdoms

Question 17

cap

Answer 17

Capping involves the addition of a methyl group to the 5’-end of the transcribed RNA
The methylated cap provides protection against degradation by exonucleases
It also allows the transcript to be recognised by the cell’s translational machinery (e.g. nuclear export proteins and ribosome)

Question 18

tail

Answer 18

at the 3’ end of the mRNA chain, a tail of 100-200 adenine molecules in a row are added (this is called poly-A)- this improves the stability of the RNA transcript and facilitates its export from the nucleus

Question 19

what are introns?

Answer 19

intervening sequences- sequences that will not contribute to the formation of the polypeptide

Question 20

what does mRNA splicing do?

Answer 20

it increases the number of different proteins an organism can produced

Question 21

what happens during mRNA splicing?

Answer 21

before it leaves the nucleus, introns removed by sn RNPs (combine with each other to form a spliceosome) to form a mature strand of mRNA which is all capable of coding for AAs

Question 22

define translation

Answer 22

the synthesis of polypeptides on ribosomes

Question 23

describe initiation (translation)

Answer 23

1. mRNA molecule binds to small ribosomal unit at an mRNA binding site
2. an initiator tRNA molecule carrying methionine binds at the start codon, AUG
3. large ribosomal unit binds to the small one
4. initiator tRNA= in P site
   another tRNA binds to the next codon= in A site
   peptide bond formed between AAs in P and A sites

Question 24

P site and A site and E site

Answer 24

A-site (aminoacyl) is the first binding site.

P-site (for peptidyl) is the second binding site

he E-site is the third and final binding site for t-RNA in the ribosome during translation

Question 25

describe elongation (translation)

Answer 25

repeated steps:
- ribosome translocates 3 bases along the mRNA, moving the tRNA in the P site to the E site, and allowing a new tRNA to bind to next codon and occupy the vacant A site

Question 26

describe termination (translation)

Answer 26

process continues until a stop codon is reached when the free polypeptide is released.

Question 27

in what direction does translation occur

Answer 27

the movement of the mRNA strand is from the 5’ end to the 3’ end

Question 28

what follows the termination of translation?

Answer 28

disassembly of the components

Question 29

distinguish between the role of free ribosomes and the role of bound ribosomes

Answer 29

free- synthesise proteins for use primarily within the cell (ie destined for cytoplasm, mitochondria and chloroplasts)
bound to endoplasmic reticulum- synthesise proteins primarily for secretion or for use in lysosomes.

Question 30

what determines whether a protein is synthesised by a free/bound ribosome?

Answer 30

the presence of a signal sequence on the polypeptide being translated (first part). this becomes bound to a signal recognition protein that stops the translation until it can bind to a receptor on the surface of the ER.

Question 31

describe protein synthesis in prokaryotes

Answer 31

translation and transcription are coupled- translation can occur immediately after transcription in prokaryotes due to the absence of a nuclear membrane.

Question 32

what are polysomes and what do they represent?

Answer 32

structures visible in an electron microscope- they represent multiple ribosomes attached to a single mRNA molecule.

Question 33

describe replication of DNA

Answer 33

the replication of DNA is semi-conservative and depends on complementary base pairing.

Question 34

describe the first 2 steps of semi-conservative DNA replication

Answer 34

1. Hydrogen bonds holding complementary bases together are broken by DNA helicase; the two strands separate and DNA unzips
2. Topoisomerase releases the strain at the replication fork, and single stranded binding proteins keep the fork open, allowing DNA helicase to function.

Question 35

describe the middle 3 steps of semi-conservative DNA replication

Answer 35

3. RNA primase catalyses the attachment of RNA primers onto the template DNA strand, allowing DNA polymerase (III) to locate the start of the template strand and attach to it
4. Free floating nucleosides in the nucleus form hydrogen bonds with complementary, exposed bases on unzipped DNA. DNA polymerase ensures that nucleosides are joined together by phosphodiester covalent bonds.
5. As the nucleosides attach to the growing DNA strand, they lose their 2nd and 3rd phosphate groups

Question 36

what are nucleosides

Answer 36

deoxynucleoside triphosphates
- DNA molecules with 3 phosphate groups rather than 1

Question 37

describe the final 2 steps of semi-conservative DNA replication

Answer 37

6. DNA polymerase III can only add nucleotides to a DNA strand in the 5’ to 3’ direction
   LEADING STRAND (5’ to 3’)- joining of nucleotides occurs continuously
   LAGGING STRAND (3’ to 5’)- multiple RNA primers needed, strand is formed in Okazaki fragments
7. once the process on the lagging strand is done, DNA polymerase I replaces primers with DNA nucleotides and the Okazaki fragments are joined together by DNA ligase.

Question 38

describe a PCR

Answer 38

Denaturation – DNA sample is heated (~90ºC) to separate the two strands
Annealing – Sample is cooled (~55ºC) to allow primers to bind to the 2 strands
Elongation – Sample is heated to the optimal temperature for a heat-tolerant polymerase (Taq) to function (~75ºC) and extend the nucleotide chain from the primers

Question 39

give an example of the universality of the genetic code

Answer 39

E. coli bacteria were modified by transferring the genes for making human insulin to it. The insulin produced has exactly the same amino acid sequence as if the gene was being transcribed and translated in human cells.

Question 40

describe the Hershey-Chase experiment

Answer 40

Viruses (T2 bacteriophage) were grown in one of two isotopic mediums in order to radioactively label a specific viral component
Viruses grown in radioactive sulfur (35S) had radiolabelled proteins (sulfur is present in proteins but not DNA)
Viruses grown in radioactive phosphorus (32P) had radiolabeled DNA (phosphorus is present in DNA but not proteins)

The viruses were then allowed to infect a bacterium (E. coli) and then the virus and bacteria were separated via centrifugation

The larger bacteria formed a solid pellet while the smaller viruses remained in the supernatant

The bacterial pellet was found to be radioactive when infected by the 32P–viruses (DNA) but not the 35S–viruses (protein)

This demonstrated that DNA, not protein, was the genetic material because DNA was transferred to the bacteria

Question 41

what are non-coding regions of DNA?

Answer 41

regions of DNA that do not code for proteins but have other important functions (eg telomeres for protective function)
- some non-coding regions play a role in the regulation of gene expression such as enhancers and silencers

Question 42

define a variable number Tandem repeat (VNTR)

Answer 42

a short nucleotide sequence that shows variations between individuals in terms of the number of times the sequence is repeated.

Question 43

define a locus

Answer 43

the physical location of a heritable element on the chromosome

Question 44

describe DNA sequencing

Answer 44

• many copies of the DNA placed in test tubes with deoxyribonucleotides, the enzymes necessary for replication and dideoxyribonucleotides that have been labelled with different fluorescent markers
• the ddns will be incorporated into some of the new DNA but will stop replication at exactly the point where they were added
• fragments separated by length using electrophoresis
• sequence of bases analysed by comparing the colour of the fluorescence with the length of the fragment

Question 45

what is gene expression regulated by?

Answer 45

proteins that bind to specific base sequences in DNA

Question 46

give examples of proteins that regulate gene expression

Answer 46

Activator proteins bind to enhancer sites and increase the rate of transcription
Repressor proteins bind to silencer sequences and decrease the rate of transcription
Binding of proteins to promoter-proximal elements is necessary to initiate transcription

Question 47

differentiate between housekeeping genes and tissue-specific genes

Answer 47

housekeeping genes are constantly required genes
tissue specific gene- function and expression are preferred in one or several tissues/cell types

Question 48

give 4 types of gene regulation

Answer 48

transcriptional- genes turned off and on
post transcriptional- modifying the mRNA so translation produces different proteins
translational- translation can be stopped or started
post translational- the structure of proteins can be changed after they are made to change their role

Question 49

the bacterium E.coli uses glucose as a respiratory substrate. In the absence of glucose E.coli can use lactose. Explain how lactose induces the enzyme system involved in its uptake and metabolism

Answer 49

1. Lac operon; a section of E.coli’s DNA that codes for enzymes that break down lactose into b glucose and galactose
2. normally a repressor substance is bound to the operator
3. this prevents RNA polymerase from binding at the promoter, thereby preventing transcription
4. lactose binds to the repressor, changing the shape of the protein molecule and causing it to be unable to bind to the operator
5. RNA polymerase binds at the promoter, and transcription occurs (= gene is switched on)
6. lactose permeate (to allow the cells to take in lactose from the surroundings) and beta-galactosidase (to break lactose down) are produced

Question 50

what are the two types of transcriptional modification?

Answer 50

• use of repressor/activator proteins
• histone modification

Question 51

gene ‘switched off’- histone modification

Answer 51

• silent (condensed) chromatin
• methylated cytosines
• deacetylated histones
• transcription prevented

Question 52

gene ‘switched on’- histone modification

Answer 52

• active (open) chromatin
• unmethylated cytosines
• acylated histones
• transcription possible

Question 53

DNA is —– and histones are ——

Answer 53

negative; positive

Question 54

adding an acetyl group to a histone

Answer 54

neutralizes this positive charge on the histone and hence reduces the binding between histones and DNA, leading to a more open structure

Question 55

adding a methyl group to a histone

Answer 55

maintains the positive charge, making DNA more coiled and reducing transcription

Question 56

describe the function and actions of tRNA activating enzymes

Answer 56

1. a specific amino acid and ATP bind to the enzyme
2. the amino acid is activated by the hydrolysis of ATP and covalent bonding of AMP
3. The correct tRNA binds to the active site. The amino acid binds to the attachment site on the tRNA and AMP is released
4. the activated tRNA is released