Structural Basis Of Cellular Information

Question 1

The chemical nature of nucleic acids

Answer 1

1869 Friedrich Miescher discovered DNA
-Nuclein
-Nucleic acid
-PA Levine determined the structure of DNA
-3 main components are:
1. Phosphate groups
2. Sugar [with Five carbons]
3. Nitrogenous bases
-Nitrogenous bases include: Purines [AG] and Pyrimidines [CT]
#Nucleotide = a unit of sugar + phosphate grp + nitrogenous base

Question 2

Nucleotides

Answer 2

• By numbering the carbons of the base and of the sugar, we identify the chemical groups.
• Sugar count clockwise from O at top
• Prime (‘) symbol indicates that the carbon is in the sugar rather than in the base.
• Phosphate group is attached to the 5’ carbon of the sugar and the base attached to the 1’ carbon
• A free hydroxyl (-OH) is attached to the 3’ carbon

Question 3

Directionality of DNA and RNA

Answer 3

• 5’ phosphate and 3’ hydroxyl group allow DNA and RNA to form long chains of nucleotides.
• Reaction between the phosphate group of one nucleotide and Hydroxyl group of another nucleotide is dehydration synthesis [removal of water] and formation of covalent bond. Bond called PHOSPHODIESTER BOND.
• Polymers [many nucleotides] are formed and they still have a free 5’ and 3’ end. This gives each DNA and RNA directionality. Bases are always expressed in 5’ to 3’ direction.

Question 4

Chargaff’s Rule

Answer 4

A guy called Erwin Chargaff showed that DNA did not contain nucleotides that were in equal proportions.

• Not a repeating polymer
• But there is regularity. The amount of Adenine is always = the amount of Thymine
• And the amount of Guanine always equals the amount of Cytosine

1. A = T and
2. G =C
   #So, its equal proportions of Purines and Pyrimidines

Question 5

3D structure of DNA

Answer 5

-Rosalind Franklin, with X-ray crystallography. Proposed that DNA is a double helix.
-Watson and Crick (1953) used all previous works to postulate that DNA is a double helix with bases pointed inwards forming base pairs between two strands
-Strands were antiparallel – one chain running 3’-5’ and other running 5’-3’
-The diameter is always 2nm. The bases are always 0.34nm apart.
-Double helix model explained Chargaff’s results [of A=T and G=C]
-Adenine forms 2 hydrogen bonds with Thymine
-Guanine forms 3 H-bonds with Cytosine
= Therefore Adenine will always occur in same proportions with thymine in any DNA molecule –this applies to G=C as well.

Question 6

REPLICATION PROCESS

Answer 6

Replication begins at one or more sites called Replication origin

• 2 strands of DNA are replicated in opposite directions
• Helicase enzyme opens and untwists DNA duplex.
• Forms replication bubbles where DNA strands are separated.
• Actual replication occurs at Y shaped ends of replication fork
• Catalysed by DNA polymerase
• RNA primer = Constructs initial 10 sequence RNA complement
• DNA polymerase recognizes the primer and adds to it.
• RNA nucleotides replaced with DNA nucleotides
• *Replication occurs only in 5’ to 3’ direction

Question 7

Proteins and Heredity

Answer 7

• Hereditary traits are determined by the changes in protein structure
• Protein structure is determined by the sequence of amino acids that make up the protein
• Sequence of amino acids determined by the sequence of nucleotides in a particular region of the chromosome
• Eg. Sickle cell disease is a mutation that replaces single thymine with adenine at a position that codes for glutamic acid, converting to valine
• Sequence of nucleotides that determines the amino acid sequence of a protein is called a gene.
• Genes code for proteins but some code.

Question 8

Cells use RNA to make protein

Answer 8

• Experiments performed with radioactive amino acids showed that although DNA directs protein synthesis proteins are not made in nucleus.
• Polypeptides assembled on Ribosomes in Cytoplasm. Ribosomes composed of RNA and proteins –very complex.

Question 9

Types of RNA

Answer 9

Cells Contain 4 Classes of RNA
• Ribosomal RNA (rRNA)
– With proteins, make up the ribosomes
– Site of polypeptide synthesis
• Transfer RNA (tRNA)
– Transport amino acid molecules to the ribosome
– Position amino acid along growing polypeptide chain
– Smaller in size than rRNA, 40 different kinds
• Messenger RNA (mRNA)
– Long strand of RNA copied from DNA
– Passes from nucleus to cytoplasm
– Conveys information from chromosomes to ribosomes
• MicroRNA (miRNA)
– Single stranded 21-23 nucleotides regulate gene
transcription and translation

Question 10

Central Dogma

Answer 10

All organisms use the same basic mechanism of reading and expressing genes.

Question 11

DNA to RNA called transcription

Answer 11

RNA polymerase binds to a promoter

• It moves along DNA strand adding complementary RNA nucleotides to growing mRNA strand.

Question 12

RNA to Protein called Translation

Answer 12

mRNA transcript is used to direct sequence of amino acids during synthesis of polypeptides
• Occurs in ribosome which moves three nucleotides at a time
• 3 nucleotides become a codeword for amino acids that are joined in a polypeptide chain

Question 13

How genes encode proteins

Answer 13

Crick determined nature of genetic code
• Blocks of information corresponding to amino acids
• Group of [3] nucleotides called a codon. Postulated code was three nucleotides long.
• 4 different nucleotides A, G, T, C
• Two nucleotide block would code for only 16 amino
acids (42=16)
• There are 20 known amino acids.
• Three nucleotide block would code for 64 amino
acids (43=64)

Question 14

Deciphering the code

Answer 14

Determination of words of code - Nirenberg and
others 1961
• Added artificial RNA to cell-free RNA and protein
• Poly-U resulted in synthesis of polyphenylalanine
• Concluded UUU coded for phenylalanine
• Repeated for all other triplets
• 64 codons possible for only 20 amino acids
• Some amino acids coded by more than one codon

Question 15

Transcription [DNA –> RNA]

Answer 15

• The first step in gene expression is production of RNA copy of DNA encoding the gene.
• RNA polymerase is responsible for the process of transcription.
• Only one of the two strands of DNA is transcribed – template strand
• The strand not transcribed is coding strand – it is the same sequence as mRNA except Thymine is Uracil on the mRNA.
• The Coding strand is sense (+) strand and template strand is the antisense (-) strand
• Polymerase adds ribonucleotides to the growing 3’ end of RNA chain
• No primer is needed and the synthesis is from 5’ – 3

Question 16

Promoter

Answer 16

• Transcription begins at RNA binding sites called Promoter on the DNA template strand.
• Promoter is a short sequence that is not transcribed by polymerase that binds to it
• In eukaryotes DNA sequence is TATAAA called TATA box at -25 nucleotides upstream [upstream is 5’ to 3’] from where transcription starts.

Question 17

Initiation of Transcription

Answer 17

• Binding of RNA polymerase (σ subunit) to promoter [TATAAA box] is first step [remember: sigma subunit recognizes the promoter and initiates synthesis]
• -25 sequence is binding site for key protein factors causing assembly to a transcription complex
• Once bound to promoter RNA polymerase begins to unwind DNA helix -17 base pairs in length -2 turns.

Question 18

Elongation [of mRNA ]

Answer 18

• Starts with ATP or GTP-forms the 5’ end
• Grows in 5’ – 3’ end as ribonucleotides are added
• No primer is required
• Region with DNA, RNA polymerase and RNA transcript is called transcription bubble
• 12 nucleotides remain attached to DNA to stabilise growing RNA in an RNA-DNA complex.
• Transcription bubble moves down DNA at 50 nucleotides per second.
• Once bubble has passed DNA is rewound.
• RNA polymerase has no proofreading capability.

Question 19

Termination [of transcription]

Answer 19

• At the end of gene are stop sequences that cause phosphodiester bonds to cease
• Causes RNA DNA hybrid to dissociate.
• And RNA polymerase to release the DNA
• Simple stop signal is series of GC base pairs followed by AT base pairs.
• The RNA transcript forms a GC hairpin followed by four or more U ribonucleotides
• RNA polymerase stops over the UUUUs which is weakest bonds with DNA’s AAAAs
• RNA dissociates with DNA and transcription stops

Question 20

Post transcription

Answer 20

• mRNA needs to travel far from nucleus to cytoplasm.
• 5’caps – transcripts usually begin with 5’ A or G – This is removed and 5’5’ linkage forms with GTP – This protects the 5’ end from nucleases and phosphatases during its journey in the cytoplasm.
• 3’ poly A tails – the eukaryotic transcript is cleaved off at specific site often containing the sequence AAUAAA.
• Special poly-A polymerase adds 250 A ribonucleotides to the 3’ end of the transcript.
• Protects the 3’ end from degradation by nuclease.

Question 21

Translation

Answer 21

• tRNA molecules carry particular amino acids attach to mRNA on ribosomes by use of an anticodon which is complementary to the mRNA triplet
• There are 45 different kinds of tRNA
• Amino acids associate with tRNA due to activating enzymes
• Called aminacyl-tRNA synthetases –one for each amino acid
• Must correspond to each tRNA and particular amino acid
• If mRNA is a coded message, 20 activating enzymes responsible for decoding that message.

Question 22

Start and Stop [of translation]

Answer 22

No tRNA with anticodon complementary to 3 of the 64 codons
• UAA, UAG and UGA –called nonsense codons serve as Stop signals in mRNA message.
• Start signal is AUG –also methionine
• Ribosome uses the first AUG to start translation
Initiation
- Polypeptide synthesis begins with the smaller ribosome subunit attaching to the mRNA
- Protein initiating factors position a tRNA on the ribosomal surface
- Two other sites will form: the A site (aminoacyl) where successive amino acid bearing tRNAs will bind and the E site where empty tRNAs will exit.
- This complex binds to the beginning of the mRNA molecule

Question 23

Elongation of the peptide chain [in translation]

Answer 23

After initiation complex has formed large subunit of ribosome binds which exposes the mRNA codon adjacent to initiating AUG codon
• This positions mRNA for interaction with another amino acid bearing tRNA molecule
• When tRNA with appropriate anticodon appears protein elongation factors bind tRNA to mRNA to A site
• When the tRNA binds the amino acid is place next to methionine amino acid which is still bound to its tRNA which is still bound to the ribosome
• Methionine released from its own tRNA and attaches to the second amino acid by a peptide bond catalysed by peptidyl transferase

Question 24

Translocation [along the Ribosome] [A, P and E sites] [in translation]

Answer 24

• Ribosomes, after attachment of amino acid, move 3 more nucleotides along the mRNA molecule in 5’ -3’ direction guided by elongation factors
• Initial tRNA relocated to E site and ejected from ribosome
• Growing polypeptide chain repositioned at p site and exposes the next codon on the mRNA at A site
• When new tRNA appears with proper anticodon, amino acid chain is attached and ribosome shifts 3 nucleotides, and process repeats itself

Question 25

Termination [of protein synthesis]

Answer 25

• Elongation continues until chain terminating nonsense codon is encountered eg UAA
• Nonsense codons do not bind tRNA but recognized by release factors the[y] release the newly made polypeptide from ribosome.

Question 26

• Replication is Semi-conservative

Answer 26

-Watson Crick Model suggests that the basis of copying genetic information is complementary
-One strand sequence determines the partner strand
• Eg. 5’-ATTGCAT-3’ partner sequence is 3’-TAACGTA-5’
-The complementarity provides means of duplication
-Unzipped strands only need to attach new [complimentary] strands to each of the parent strands to create daughter duplexes with the same sequence. This is called semi-conservative replication

Question 27

What are the leading and lagging strands?

Answer 27

1. Leading strand = 5’ to 3’ strand : new strand grows from 3’ end ad elongates TOWARDS replication fork.
2. Lagging strand = 3’ to 5’ strand replication. Elongates away from replication fork – Synthesized discontinuously in small batches
   – 5’ to 3’ synthesis catalysed by DNA polymerase
   • Segments called Okazaki fragments
   • DNA ligase attaches fragment to lagging strand
   • Overall replication process is termed semidiscontinuous

Question 28

RNA polymerase in bacteria

Answer 28

> In bacteria - [RNA polymerase] consists of 5 subunits:
2 Alpha subunits bind regulatory proteins
1 Beta’ subunit binds the DNA template
1 Beta unit binds RNA nucleoside subunits
1 Sigma subunit recognises the promoter and initiates synthesis.

Question 29

There are 3 different polymerases in eukaryotes:

Answer 29

• > • RNA pol I – synthesizes rRNA
• > • RNA pol II synthesizes mRNA
• > • RNA pol III synthesizes tRNA