Central Dogma

I. Overview of the Central Dogma

The central dogma describes the flow of genetic information from DNA to functional proteins.

• Transcription: Occurs in the nucleus; DNA is used as a template to synthesize mRNA.

• Translation: Occurs in the cytosol at the ribosome; mRNA is read to synthesize proteins.

DNA  →  RNA  →  Protein

This is the fundamental principle of molecular biology. Genetic information flows from DNA to RNA (transcription) and then from RNA to protein (translation). The original DNA remains intact and is protected in the nucleus, while working copies (mRNA) carry instructions to the cytoplasm.

TRANSCRIPTION

What is Transcription?

• The copying of the genetic code from DNA to RNA

• Involves synthesis of RNA using DNA as a template

• Major enzyme: RNA Polymerase

• Ensures genetic information is faithfully reproduced while original DNA stays intact and protected from alteration or degradation

Three Types of RNA

II. Transcription: From DNA to RNA

Transcription is the process of copying the genetic code from DNA into RNA while keeping the original DNA protected from degradation.

Key Characteristics:

• Template Strand: Only one DNA strand (the 3' to 5' strand, also called the antisense strand) is transcribed. The non-template strand is called the sense strand or coding strand (5' to 3')

• Base Pairing: C pairs with G, and A pairs with U (Uracil replaces Thymine).

• mRNA Product: The resulting mRNA is a copy of the (non-template) sense strand (5' to 3'), with U substituted for T.

Requirements & Enzyme Structure

1. Gene (parts of the genome or entire DNA): The part of the DNA coding for a functional protein.

• Segment of the genome (entire DNA) that codes for a functional protein

• The gene has three parts:

• Promoter: Where RNA polymerase binds to initiate transcription

• RNA-coding sequence: The region actually transcribed

• Terminator: Signals end of transcription

• Numbering convention: first base in RNA = +1; bases before that = -1, -2, -3...

2. Nucleotide Triphosphates: 

• ATP (Adenosine triphosphate)

• CTP (Cytidine triphosphate)

• GTP (Guanosine triphosphate)

• UTP (Uridine triphosphate)

3. RNA Polymerase: The major enzyme responsible for synthesis. recognizes It has 5 subunits:

Steps in Transcription

1. Initiation

• RNA polymerase binds to the Promoter region

• Starts at the -35 region, then migrates to the -10 region (Pribnow box) to form an open complex

• The sigma (σ) factor recognizes the promoter sequence — runs from approximately -70 to +1 (the transcription start site)

• Consensus sequences: -35 box (recognition site) and -10 box (sigma box/unwinding site)

• Open complex forms → DNA strands unwind

• Conformational change converts the complex to the elongation form

• RNA polymerase activates and begins synthesis

• After 8–9 nucleotides are synthesized → sigma factor dissociates from RNA polymerase

• 5' TATAAT 3' → -10 promoter sequence

2. Elongation

• NusA protein associates with RNA polymerase after sigma dissociates → signals RNA polymerase for elongation and helps regulate pausing

• RNA synthesis is processive → continuously adds nucleotides without detaching from the DNA template

• If RNA polymerase pauses, termination can begin

3. Termination

Two mechanisms in E. coli:

Mechanism 1—Rho-Independent (Intrinsic/Hairpin Termination):

• The RNA transcript contains self-complementary (palindromic) sequences—these read the same on both strands in antiparallel orientation, allowing the single RNA strand to fold back and base-pair with itself

• This forms a hairpin loop structure at 15–20 nucleotides

• The hairpin is stabilized by intramolecular hydrogen bonding

• Rho-independent termination is characterized by a GC-rich hairpin followed by a poly-U tail

• The hairpin causes disruption → dissociation of RNA from the DNA template → termination begins

• RNA polymerase reaches a termination signal in the DNA

• This signal codes for an RNA sequence that folds back on itself to form a HAIRPIN structure

• The hairpin causes the RNA strand to separate from RNA polymerase, ending transcription

• palindromic sequences in DNA form a hairpin structure (also known as a stem-loop) in the newly synthesized RNA that signals the termination of transcription in prokaryotes, specifically in a process called Rho-independent or intrinsic termination.

Mechanism 2—Rho-Dependent Termination:

• The Rho protein binds to the rut element (Rho Utilization site) on the RNA

• Rho protein is an ATP-dependent helicase (RNA helicase)

• RNA helicase is activated by hydrolysis of ATP

• The rut element is cytosine-rich

• Rho moves along the RNA in the same direction as RNA polymerase

• Rho catches up to the paused polymerase and pulls the RNA away → termination

• Rho protein (an ATP-dependent helicase) binds to the RNA

• Rho uses ATP hydrolysis to pull RNA away from the polymerase and template

• This physically dislodges the RNA and terminates transcription

mRNA & POST-TRANSCRIPTIONAL PROCESSING

• Eukaryotic primary transcript (pre-mRNA) is initially non-functional and requires processing before it can be used

• Prokaryotic mRNA is directly functional — no processing needed because transcription and translation are coupled

Post-Transcriptional Processing — Eukaryotes ONLY

Three stages:

1. Splicing

• The initial mRNA transcript (pre-mRNA) contains both coding (exon) and non-coding (intron) sequences

• The spliceosome (composed of snRPs — small nuclear ribonucleoproteins) removes the introns

• Exons are joined together to form mature mRNA

• Removal of introns (non-coding ribonucleotide sequences)

• Carried out by the spliceosome

• Spliceosome (protein) is made up of snRNPs (small nuclear ribonucleoproteins, pronounced "snurps")

• Splicing joins the exons together → gives a complete, uninterrupted coding sequence for protein synthesis

• Eukaryote has multiple origins of replication because of A-T base pairs

2. 5' Capping

• A 7-methylguanosine (m⁷G) cap is added to the 5' end of the mRNA

• The 5' cap serves as the binding site for the ribosome during translation initiation

• The cap also protects the mRNA from degradation by ribonucleases

3. 3' Poly-A Tailing

• The 3' end is cleaved and undergoes polyadenylation

• A string of adenine (A) residues (approximately 100–300 nucleotides) is added to the 3' end

• Protects the mRNA from nuclease degradation

• A 7-methylguanosine (m7G) cap is added to the 5' end

• A poly-A tail (a long string of adenines) is added to the 3' end

• Both modifications protect the mRNA from nucleases (degradation enzymes) when it exits the nucleus

• Structure of mature eukaryotic mRNA: 5' cap — Coding Region — Poly(A) tail 3'

Mature mRNA

• After all three processing steps, the mature mRNA travels: nucleus → cytosol → ribosome

• At the ribosome, translation begins

III. Types of RNA

IV. Post-Transcriptional Processing

Before mRNA leaves the nucleus, it must be modified:

1. Splicing: Removal of introns (non-coding) and joining of exons (coding).

2. 5' Capping: Adding a 7-methylguanosine "cap".

3. 3' Poly-A Tailing: Adding a tail of Adenines to prevent degradation.

V. Translation: From RNA to Protein

Translation uses mRNA codons to determine the specific order of amino acids in a polypeptide.

• The process by which the mRNA sequence is decoded to build a specific protein

• The mRNA sequence (codons) is read and matched to specific amino acids

• Takes place at the ribosome in the cytoplasm

• Direction: mRNA is read 5' to 3'

• mRNA sequence forms a message (order of amino acids in a polypeptide)n

• message translation: mRNA CODONS → specific amino acids

The Genetic Code

• Codon: A sequence of 3 consecutive ribonucleotides (triplet code).

• Each codon is specific for one amino acid except for stop codons.

• Total possible codons = 4³ = 64

• Number of amino acids = 20

• One amino acid can be coded by 1 to 6 codons (the code is degenerate)

• Start Codon: AUG (codes for Methionine).

• Stop Codons: UAG, UGA, UAA.

• Degenerate: Most amino acids are coded by more than one codon.

Features of the Genetic Code

• Non-overlapping: codons are read in successive, non-overlapping groups of 3

• Degenerate: one amino acid may be coded by more than 1 codon

• Universal: the same genetic code is used by virtually all organisms on Earth

• Has START and STOP codons:

• START codon: AUG (codes for Methionine, Met) — every protein begins with Met

• STOP codons: UAG, UGA, UAA — no amino acid; signal end of translation

Amino Acid Codon Summary

Requirements for Translation

The ribosome (70S in Prokaryotes, 80S in Eukaryotes) has three sites:

• A-site: Aminoacyl-tRNA binding (the "entrance").

• P-site: Peptidyl-tRNA binding (where the peptide chain grows).

• E-site: Exit site.

Steps of Translation

1. Activation: Aminoacyl-tRNA synthetase attaches the correct amino acid to tRNA.

2. Initiation: Small subunit binds to mRNA (Shine-Dalgarno sequence in prokaryotes); first tRNA binds to AUG.

3. Elongation: tRNAs bring amino acids; peptidyl transferase forms bonds; translocation moves the ribosome along the mRNA.

4. Termination: A Release Factor binds to the stop codon in the A-site, releasing the completed protein.

Template: Types of mRNA

Prokaryotic mRNA

• Has a Shine-Dalgarno sequence — the ribosomal binding site upstream of the start codon

• Polycistronic: one mRNA can encode MULTIPLE proteins

• No 5' cap or poly-A tail (no post-transcriptional processing in prokaryotes)

Eukaryotic mRNA

• Monocistronic: one mRNA encodes only ONE protein

• Has a 5' methylguanosine cap and 3' poly-A tail

• mRNA must exit the nucleus through nuclear pores to reach ribosomes in the cytoplasm

The Ribosome

• Site of translation — where mRNA is read and protein is built

• Consists of two subunits: small subunit and large subunit

• Contains three important sites:

• A site (Aminoacyl-tRNA binding site): where the incoming charged tRNA (carrying a new amino acid) binds

• P site (Peptidyl-tRNA binding site): where the growing polypeptide chain is held and where amino acid has transferred

• E site (Exit site): where the empty or uncharged tRNA exits the ribosome
  

tRNA Structure

• Acts as the adaptor molecule that bridges codons and amino acids

• Transports cystolic AAs to ribosome

• Self-complementary bases

• hairpin

• left and right arm is for the stability

• 5’ attachment of AA

• binds to mRNA by complementary base pair codon

• 3’5 -5’ to 5’-3’ mRNA

• each AA has specific tRNA which comprises 40-60 types of tRNA

• Has a CLOVERLEAF structure formed through intramolecular hydrogen bonding

• Upper end (3' end): amino acid attachment site — where the specific amino acid is loaded

• Lower end: anticodon loop — contains the 3-base anticodon that base-pairs with the mRNA codon

• Anticodon is complementary and antiparallel to its corresponding mRNA codon

Steps in Translation

Step 1: Activation of tRNA with Amino Acids

• Each tRNA must be charged (loaded) with its specific amino acid before translation begins

• Enzyme responsible: Aminoacyl-tRNA Synthetase (hydrolysis of ATP)

• Mg2+ dependent

• Reaction uses ATP: Amino acid + ATP → Aminoacyl-AMP → Aminoacyl-tRNA + AMP

• This ensures the correct amino acid is attached to the correct tRNA

• tRNA^Ala tRNA ^Asp tRNA^Gln tRNA^Ser

Step 2: Initiation — Formation of the Initiation Complex

• (a) Initiation Factors (IFs) bind to the Shine-Dalgarno sequence (prokaryotes) and recruit the small ribosomal subunit

• (b) The initiator tRNA (carrying Methionine/Met) binds to the AUG start codon at the P site

• (c) GTP hydrolysis causes the IFs to dissociate from the complex

• (d) The large ribosomal subunit joins to complete the 70S (prokaryote) or 80S (eukaryote) initiation complex

• 5’ cap added to association of auxillary proteins (IF123s)

• looks for start codon

• small ribosomal unit will pause at AUG

• Powered by GTP hydrolysis

• Dissociate where large ribosomal forms initiator complex between small and large subunits

Step 3: Elongation (repeating cycle)

steps 1,2,3 and formula

• (a) A new aminoacyl-tRNA enters the A site — its anticodon must match the codon in the A site

• (b) Peptide bond formation: the growing polypeptide chain (held at P site) is transferred to the amino acid at the A site — catalyzed by PEPTIDYL TRANSFERASE (an rRNA enzyme — ribozyme)

• (c) Translocation: the ribosome moves one codon (3 bases) in the 3' direction:

• tRNA from A site moves to P site (now carrying the growing chain)

• tRNA from P site moves to E site

• tRNA exits from E site

• A site is now vacant and ready for the next aminoacyl-tRNA

• (d) Cycle repeats until a stop codon enters the A site

• P-site tRNA cleaves to mRNA (peptide)

• move 1 codon to 3’ end (translocation) until it reads stop codon

• UAA UAG → RF1 and RF3

• UAA UGA -. RF2 AND RF3

• hydrolysis → polypeptide from tRNA followed by folding

Step 4: Termination

• A stop codon (UAG, UGA, or UAA) enters the A site

• No tRNA recognizes stop codons — instead, a RELEASE FACTOR binds to the A site

• The release factor activates peptidyl transferase to add water (H₂O) instead of an amino acid — this hydrolyzes the bond between the polypeptide and the last tRNA

• The completed polypeptide (protein) is released

• The ribosomal subunits dissociate and can be reused

PART 3: QUICK REFERENCE TABLES

Transcription vs. Translation

Prokaryote vs. Eukaryote (Transcription & Translation)

RNA Polymerase Subunit Summary

PART 4: KEY TERMS GLOSSARY

PART 5: EXAM TIPS & COMMON PITFALLS

Common Mistakes to Avoid

• Don't confuse the TEMPLATE strand with the CODING (sense) strand. The mRNA sequence matches the CODING strand (except U for T).

• Don't confuse TRANSCRIPTION (DNA→RNA) with TRANSLATION (RNA→Protein).

• Don't say RNA polymerase uses DNA primers — it doesn't need one, unlike DNA polymerase.

• Don't confuse ANTICODON (on tRNA) with CODON (on mRNA). They are complementary and antiparallel.

• The sigma factor is released AFTER initiation — it's only needed to find the promoter.

• In eukaryotes, transcription and translation are SEPARATED in space (nucleus vs. cytoplasm). In prokaryotes, they are COUPLED.

Good luck on your exam!