Chapter 6: How Cells Read the Genome -> From DNA to Protein

As interesting as it sounds the DNA in genomes doesn’t direct the protein synthesis on its own. It uses RNA as the middleman to do the job.

As we deep dive into concepts, we will explore how genetic information flows from DNA → RNA → Protein. This is often referred to as the Central Dogma of Molecular Biology.

The 4 different nucleotides in DNA (A, T, G, C) directs the formation of human, fruit fly, or bacteria. It’s crazy what these four letters when compiled can do.

RNA (Ribonucleic Acid)

• Ribose sugar is present in RNA.
• Uracil is present instead of thymine.
• RNA is single-stranded.
• “Although these chemical differences are slight, DNA and RNA differ quite dramatically in overall structure”.
• Major types of RNAs:
• t-RNA: Transfer RNA; at the time of protein synthesis, it acts as a carrier of amino acids.
• mRNA: Messenger RNA; codes for proteins.
• r-RNA: Ribosomal RNA; form the basic structure of the ribosome and catalyze protein synthesis.

MECHANISM OF DNA REPLICATION:

• Steps:
• Initiation of DNA replication: The initiation starts with the ORI which in eukaryotes is many origins of replication whereas in prokaryotes it is a single origin of replication.
• The unwinding of DNA: The DNA strands unwind with the help of DNA helicase.
• Removal of supercoiling: Topoisomerase in eukaryotes which is also DNA gyrase in prokaryotes releases the tension that arises due to supercoiling.
• Formation of new strands: To start forming new strands, RNA primers are catalyzed by RNA primase. Synthesis takes place in the direction of 5’ → 3’. As a result, one chain of DNA is called the leading strand. The discontinuous chain is called the lagging strand. The small segments are called Okazaki fragments.
• Proof Reading: Once the initial primer is removed from DNA polymerase I go for proofreading and add the nucleotides in the correct sequence.
• Ligation: DNA ligase joins the Okazaki fragments.

TRANSCRIPTION:

• It is the process in which genetic information is copied from one DNA strand to one RNA strand.
• RNA polymerase which is involved in transcription is basically of three types:
• RNA Polymerase I: 28 s-rRNA, 18 s-rRNA, 5.8 s-rRNA
• RNA Polymerase II: hnRNA → mRNA
• RNA Polymerase III: t-RNA, 5 sRNA, and snRNA (small nuclear RNA, helps in RNA splicing and also help in the formation of spliceosomes.
• For transcription, the total DNA of a cell does not participate in the process, a part of DNA goes for transcription.
• Only one strand goes for transcription which is the template strand.
• It also follows the complementarity rule.
• formation of m-RNA (5’ → 3’).
• Transcription unit:
• Promoter: RNA Polymerase binding site
• Terminator: Transcription stop site
• Structural gene: It is the actual RNA coding region
• Requirements:
• DNA Template
• RNA Polymerase
• Ribonucleotides (ATP, CTP, GTP, UTP)
• RNA Polymerase:
• It cut hydrogen bonds
• It also reads the template strand
• The formation of new m-RNA in the 5’ → 3’ direction by using appropriate nucleotide.
• Structure of RNA Polymerase:
• DNA-Dependent RNA Polymerase
• Molecular Weight: 465 Dalton
• It is made up of six polypeptides (2 alpha, beta, beta dash, omega, and sigma factor)
• Core Enzyme + sigma factor = RNA Polymerase
• Alpha assists the sigma factor for correct binding.
• Beta and Beta dash helps in the unwinding of DNA.
• Steps for transcription:
• Initiation:
  • DNA has a promoter site where RNA polymerase binds and a terminator site where transcription stops.
  • Sigma factor recognizes the promoter site of DNA.
  • With the help of sigma factor RNA polymerase enzyme is attached to a specific site of DNA called “promoter site”.
  • In prokaryotes before the 10 N-bases from starting point, a sequence of 6 base pairs (TATAAT) or (TATATAT) is present in DNA, which is called the Pribnow box.
  • In eukaryotes before the 20 N-bases from starting point, a sequence of 7 base pairs (TATAAAA) OR (TATATAT) is present in the DNA which is called TATA box or Hogness box.
  • At the start point, the RNA polymerase enzyme breaks the H-bonds of two strands of DNA and separates them.
  • One of the strands takes part in transcription. Transcription proceeds in a 5’ -→ 3’ direction.
  • Ribonucleoside triphosphates come to lie opposite of complementary nitrogen bases of the antisense strand.
  • These ribonucleotides are present in the form of triphosphate ATP, GTP, UTP, and CTP. When they are used in transcription, the pyrophosphatase enzyme hydrolyzes two phosphates from each NTP (Triphosphate), which releases energy. This energy is used in the process of transcription.
• Elongation:
  • RNA Polymerase enzyme establishes phosphodiester bonds between adjacent ribonucleotides.
  • Sigma factor separates and RNA polymerase moves along the anti-sense strand till it reaches the terminator site.
• Termination:
  • When the RNA polymerase enzyme reaches the terminator site, it separates from the DNA template.
  • In prokaryotes, the terminator site is recognized with the help of the Rho factor.
  • Rho factor is a specific protein that helps in termination.
  • Inheritance of a character is also affected by promoter and regulatory sequences of a structural gene. Hence, sometimes helps the regulatory sequences are loosely defined as regulatory genes, even though these sequences do not code for any RNA or protein.

TRANSLATION:

• Polymerization of amino acid.
• Chain of nucleotides → Chian of amino acids.
• Site: Cytoplasm
• Requirements:
• 20 standard amino acids.
• mRNA
• tRNA
• ribosome
• Mg+2
• GTP/ATP
• translation factors
  • initiation process
  • elongation process
  • termination process
• Mechanism:
• Activation of amino acids.
• Amino acid + ATP → (aminoacyl t-RNA synthetase) Amino acyl AMP enzyme complex + Pi
• Charging of t-RNA (loading of t-RNA.
• Aminoacyl AMP enzyme complex (charged t-RNA)+ t-RNA -→ aminoacyl + RNA complex + AMP
• Steps:
• Initiation:
  • In this step 30s and 50s sub-units of ribosome, GTP, Mg+2, charged t-RNA, m-RNA, and some initiation factors are required.
  • In prokaryotes there are three imitation factors present- IF1, IF2, IF3
  • Initiation factors are specific proteins.
  • GTP and initiation factors promote the initiation process.
  • In prokaryotes with the help of the “S D sequence” m-RNA recognizes the smaller subunit of ribosomes. A sequence of few N-bases is present before the 4-12 N-bases of initiation codon on m-RNA, called SD sequence is present on 16s rRNA.
  • With the help of SD and ASD sequences, mRNA and the smaller sub-unit of the ribosome are attached.
  • While in eukaryotes, a is the smaller sub-unit protein of ribosome is recognized by “7mG cap”.
  • In eukaryotes, 18s r-RNA of smaller sub-unit has a complementary sequence of “7mG cap”.
  • This 30s-mRNA complex" reacts with formyl methionyl t-RNA complex and 30s-mRNA-formyl methionyl t-RNA complex is formed. This t-RNA attaches to the codon part of m-RNA. A GTP molecule is required.
  • Now larger subunit of ribosome joins this complex. The initiation factor is released and a complete 70s ribosome is formed.
  • In larger subunits of ribosome there are three sites for t-RNA:
  • P site = Peptidyl site
  • A site = Amino acyl site
  • E site = Exit site
  • Start codon of m-RNA is near to P site of the ribosome, so t-RNA with formyl methionine amino acid first attaches to the P site of the ribosome, and the next codon of m-RNA is near to A-site of the ribosome. So the next new t-RNA with new amino acid always attaches at the A site of the ribosome but in the initiation step, the A site is empty.
• Elongation:
  • New t-RNA with new amino acid is attached at the A site of the ribosome.
  • The link between amino acid of Psite t-RNA is broken and t-RNA of P-site is discharged so -COOH of P-site A. A becomes free.
  • Now peptide bond formation takes place between the -COOH group of P-site amino acid and the -NH2 group of A-site amino acid.
  • 23 s-r-RNA induces the formation of P site released from ribosomes via E-site and dipeptide attaches with A-site.
  • Now t-RNA of A site is transferred to P-site and the A site becomes empty.
  • Now ribosome slides over m-RNA strand in 5’ -→ 3’ direction. Due to the sliding of ribosome on m-RNA, a new codon of m-RNA is continuously available at the A site of the ribosome and according to the new codon of m-RNA new amino acid attaches in a polypeptide chain.
  • Translocase enzyme is helpful in the movement of the ribosome, GTP provides energy for sliding of the ribosome.
  • In the elongation process some protein factors are also helpful, which are known as elongation factors.
  • In prokaryotes three elongation factors are present -EF-Tu, EF-Ts, EF-G
• Termination:
  • Due to the sliding of ribosomes over m-RNA when any nonsense codon (UAA, UGA, UAG) becomes available at the A site of ribosomes, then the polypeptide chain terminates.
  • The linkage between the last t-RNA and the polypeptide chain is broken by three release factors called RF1, RF2, and RF3 with the help of GTP.
  • At the end, a release factor binds to the stop codon, terminating translation and releasing the complete polypeptide from the ribosome.
  • An mRNA also has some additional sequences that are not translated and are referred to as untranslated regions (UTR). The UTRs are present at both 5’end (before the start codon) and at 3’end (after the stop codon).
  • The UTR present in mRNA is required for an efficient translation process by recognizing the smaller subunit of the ribosome by mRNA.