Micro exam 3 central dogma

Central Dogma of Molecular Biology

The central dogma describes the directional flow of genetic information — [DNA → RNA → Protein]. DNA stores hereditary information, RNA carries the message through transcription, and proteins perform cellular functions through translation.

Gene Expression in Microbes

Gene expression converts genetic code into functional molecules — mRNA, rRNA, tRNA, or protein. Microbes depend on gene expression for metabolism, structure, and adaptation to environments.

Structural Gene

A DNA segment coding for a protein or functional RNA; contains a coding region plus regulatory sequences (promoter, leader, terminator) that control transcription and translation.

Coding Region

The part of a gene transcribed and translated into amino acids; begins at start codon (AUG) and ends at stop codon (UAA/UAG/UGA).

Genetic Map Positions (0, +1, −1)

The transcription start site is +1; nucleotides downstream are positive (+2, +3…), upstream are negative (−1, −2…). This system orients genetic features [Upstream ← 0 → Downstream].

Upstream vs Downstream

Upstream = toward 5′ end (DNA template); downstream = toward 3′ end. Promoters lie upstream; coding regions extend downstream.

Promoter

DNA region upstream of a gene where RNA polymerase + σ factor bind to start transcription. Typical bacterial promoter: [−35 box TTGACA] … [−10 box TATAAT] → Start (+1).

Sigma Factor (σ)

Protein that guides RNA polymerase to specific promoter motifs; ensures correct gene selection. Multiple σ factors allow environmental regulation (e.g., heat shock σ³²).

Sigma-70 (σ⁷⁰)

The “housekeeping” sigma factor used by most genes under normal growth; recognizes consensus −10/−35 sequences and initiates transcription of essential genes.

Closed Complex

Initial promoter–RNA polymerase holoenzyme binding; DNA strands remain paired — enzyme is “positioned” but not yet open.

Open Complex

After promoter melting at the −10 region, DNA strands separate and RNA polymerase synthesizes the first ~12 nt of RNA — σ factor soon dissociates. [Closed → DNA melts → Open → σ release]

Leader (5′ UTR)

Non-translated RNA upstream of start codon; includes ribosome-binding site (Shine–Dalgarno) and controls translation efficiency.

Shine–Dalgarno Sequence

Purine-rich (AGGAGG) region ~7 nt upstream of start codon that base-pairs with 16S rRNA in 30S subunit → positions ribosome for correct initiation.

Start Codon (AUG)

Signals translation initiation; codes for formyl-methionine (fMet) in bacteria.

Trailer (3′ UTR)

mRNA region following coding sequence containing termination signals and regulatory elements affecting RNA stability.

Terminator

DNA sequence that ends transcription; forms a stop signal for RNA polymerase. Can function via intrinsic (ρ-independent) or ρ-dependent mechanisms.

Intrinsic Termination

Occurs when transcribed RNA forms a GC-rich stem-loop followed by U-rich tract — weak U:A pairing → mRNA pulls free from RNAP. [Stem-loop ⊣ U-tract → Release]

Rho-Dependent Termination

Requires ρ protein (ATPase + helicase). ρ binds rut site on RNA, moves 5′→3′ using ATP, catches RNAP → pulls RNA out. [rut → ρ binds → ATP → Pull → Stop]

rut Sequence

C-rich, unstructured region in mRNA that serves as binding site for ρ protein in ρ-dependent termination.

Operon

Cluster of functionally related genes controlled by one promoter and transcribed as a single polycistronic mRNA. [Promoter → Gene A → Spacer → Gene B → Gene C]

Polycistronic mRNA

One mRNA containing multiple cistrons (each with start + stop codon); enables coordinated expression of related proteins (e.g., lac operon).

Monocistronic mRNA

mRNA encoding only one protein (product of a single gene); common in eukaryotes and some bacterial genes.

Cistron

Functional unit within polycistronic mRNA bounded by start/stop codons; produces one polypeptide chain.

Spacer Sequence

Noncoding region between cistrons; may affect ribosome reinitiation or translation rate of downstream gene.

RNA Polymerase Core Enzyme

Catalytic complex (α₂ββ′ω) that synthesizes RNA but needs σ factor for promoter recognition. Core alone = “blind transcription.”

RNA Polymerase Holoenzyme

Core enzyme + sigma factor → forms initiation-competent complex able to recognize promoters. [α₂ββ′ω + σ → Holoenzyme]

Phases of Bacterial Transcription

Initiation: σ binds promoter → open complex. Elongation: σ releases; RNAP adds NTPs. Termination: RNAP stops at terminator. [Init → Elong → Term]

Intake Port / Nucleotide Uptake Channel / Exit Channel

RNAP features for DNA entry, NTP entry, and mRNA exit. DNA unwinds at intake, NTPs enter active site, mRNA leaves exit port.

Burst-and-Pause Elongation

RNAP moves in rapid bursts (100–200 nt) separated by pauses. Pauses allow RNA folding or regulatory protein binding.

Transcription Regulation by Pausing

Temporary halting of RNAP can expose attenuators or riboswitches that control whether transcription continues or terminates.

Prokaryotic vs Eukaryotic mRNA Structure

Prokaryotic mRNA: no introns, no 5′ cap, no poly-A tail, often polycistronic, translated in cytoplasm. Eukaryotic mRNA: introns removed (splicing), 5′ cap + 3′ poly-A tail, monocistronic, exported from nucleus.

Cotranscriptional Translation

In bacteria, ribosomes bind nascent mRNA while it is still being synthesized. [DNA → RNAP → mRNA → Ribosomes]

Advantage of Cotranscriptional Translation

Allows rapid response to environmental changes and reduces mRNA degradation time — critical for fast-growing microbes.

Promoter Strength

Determined by how closely −10 and −35 motifs match consensus and their spacing. Strong promoters → frequent transcription.

Alternative Sigma Factors

Specialized σ proteins (heat shock, sporulation, nitrogen starvation) redirect RNAP to new promoter sets for adaptive gene expression.

Leader → Coding → Trailer → Terminator

Full gene organization in bacteria — [Promoter → Leader → Coding → Trailer → Terminator]. Only leader, coding, trailer are transcribed into mRNA.

Role of Stem-Loop Structures in RNA

Hairpins can terminate transcription, block ribosome binding, or act as regulatory elements in riboswitches responding to metabolites.

mRNA Stability Differences

Bacterial mRNAs short-lived (minutes) due to RNases and lack of protective cap/tail; eukaryotic mRNAs stable (hours) via 5′ cap and poly-A tail.

Why Prokaryotic mRNAs Need Minimal Modification

Transcription and translation occur in cytoplasm → no need for export signals; rapid gene expression outweighs long-term stability.

Role of fMet-tRNAᶠᴹᵉᵗ

Special initiator tRNA carrying N-formylmethionine pairs with AUG start codon; formyl group marks beginning of polypeptide chain in bacteria.

Transcription vs Translation Coupling

Because ribosomes bind nascent mRNA quickly, transcription and translation are functionally linked — gene expression can be regulated by ribosome progress.

RNAP Pausing and Attenuation

Pauses allow leader peptides or RNA structures to determine whether transcription continues — classic example: trp operon attenuation.

Terminator vs Trailer Distinction

Trailer = region of mRNA near 3′ end with signals for termination or regulation. Terminator = specific DNA sequence that stops RNAP.

ρ Protein Activities

Has dual ATPase (energy for movement) and Helicase (unwraps RNA) functions. Converts chemical energy into mechanical force to detach RNA from RNAP.

Upstream Activators and Promoter Enhancement

Certain proteins bind upstream of −35 region to bend DNA and facilitate RNAP recruitment — increasing transcription rate even if core motifs are weak.

RNAP vs DNA Polymerase Difference

RNAP does not require a primer and uses NTPs (not dNTPs); DNA polymerase needs a primer and only copies DNA during replication.

Introns and Splicing in Microbes

Rare in bacteria/archaea (but exist in tRNA genes of archaea); common in eukaryotes → must be spliced out before translation.

Genetic Economy of Operons

By grouping genes under one promoter, bacteria save energy and coordinate expression of pathway enzymes (e.g., lac, trp, ara operons).

Transcriptional Polarity

A mutation causing premature termination in an upstream cistron can reduce translation of downstream genes in the same operon (due to loss of ribosome coverage).

Central Dogma Summary Diagram

[DNA (replication) → Transcription → mRNA → Translation → Protein → Function] Emphasizes the flow and regulation of genetic information in microbial cells and how transcriptional control determines phenotype.

1. What is the purpose of transcription?

To create an mRNA copy of the coding region of a gene or operon, allowing genetic information in DNA to be expressed as protein.

2. In what direction is RNA synthesized?

RNA is synthesized 5′ → 3′, meaning RNA polymerase reads the template DNA strand 3′ → 5′.

3. What are the base-pairing rules for RNA transcription?

G pairs with C, and A pairs with U.

4. Which DNA strand serves as the template during transcription?

The template strand; the resulting mRNA is complementary to this strand and identical (except U replaces T) to the sense (coding) strand.

5. What is the leader sequence (5′-UTR) in mRNA?

A short noncoding region before the start codon that helps the ribosome initiate translation.

6. What is a codon?

A group of three nucleotides on mRNA that corresponds to one amino acid.

7. What is the start codon and its role?

AUG, which codes for N-formyl-methionine (fMet); it establishes the reading frame for translation.

8. What are the three stop codons and their nicknames?

UAG (amber), UAA (ochre), UGA (opal) — each signals termination of translation.

9. What does “reading frame” mean?

The correct grouping of mRNA nucleotides into triplets (codons) that determines the resulting amino acid sequence.

10. What molecule translates codons into amino acids?

Transfer RNA (tRNA) molecules, each carrying a specific amino acid corresponding to its anticodon.

11. What is the structure of tRNA?

A single-stranded RNA that folds into a 3D cloverleaf shape via internal base pairing.

12. What are the four functional regions of tRNA?

13. What is the anticodon loop?

The tRNA region containing a triplet complementary to the mRNA codon.

14. What is the acceptor stem?

The site where the correct amino acid is covalently attached.

15. What enzyme attaches amino acids to tRNA?

Aminoacyl-tRNA synthetase, which uses ATP to link the amino acid to its corresponding tRNA.

16. How does aminoacyl-tRNA synthetase ensure accuracy?

It recognizes both the anticodon loop and D-arm of the correct tRNA before attachment.

17. What is an aminoacyl-tRNA?

A tRNA molecule with its correct amino acid attached at the acceptor stem.

18. What is the bacterial ribosome’s total sedimentation coefficient?

70S, composed of 50S and 30S subunits.

19. What are the rRNA molecules in the bacterial ribosome? |

• 50S: 5S rRNA and 23S rRNA

• 30S: 16S rRNA

20. What is “Svedberg” (S) a measure of?

Sedimentation rate — how fast a molecule travels during ultracentrifugation; it reflects size and shape, not exact weight.

21. What are the three tRNA binding sites in the ribosome?

A-site (Aminoacyl site), P-site (Peptidyl site), E-site (Exit site).

22. What happens at the A-site?

The incoming aminoacyl-tRNA binds to the ribosome.

23. What happens at the P-site?

A peptide bond forms between the amino acids in the A-site and P-site.

24. What happens at the E-site?

The now-empty tRNA exits the ribosome.

25. What are the three main stages of bacterial translation?

Initiation, Elongation, and Termination.

26. What components are required for translation initiation?

The 30S subunit, the mRNA, initiator tRNA (fMet-tRNA), and three initiation factors (IF-1, IF-2, IF-3).

27. What is the Shine-Dalgarno sequence?

A purine-rich region in the mRNA leader that base-pairs with 16S rRNA to align the start codon in the P-site.

28. What is the function of IF-3?

Binds to the 30S subunit to prevent premature joining with the 50S subunit.

29. What does IF-2 do?

Binds GTP and the initiator fMet-tRNA, helping position it at the start codon.

30. What is the role of IF-1?

Binds the 30S subunit after fMet-tRNA pairing, causing IF-3 to release — forming the 30S initiation complex.

31. What event marks completion of initiation?

The 50S subunit joins the 30S complex, and IF-2 hydrolyzes GTP, locking both subunits together into the 70S initiation complex.

32. Diagram: Stepwise assembly of bacterial translation initiation |

33. What are the three repeating steps of elongation?

Aminoacyl-tRNA binding, Transpeptidation, and Translocation.

34. What elongation factor is used in aminoacyl-tRNA binding?

EF-Tu, which delivers tRNA to the A-site using GTP hydrolysis.

35. What happens during transpeptidation?

The 23S rRNA (ribozyme) of the 50S subunit catalyzes a peptide bond between amino acids in the A- and P-sites.

36. What occurs during translocation in elongation?

The entire ribosome shifts one codon downstream on the mRNA; the tRNA in the A-site moves to the P-site, the P-site tRNA moves to the E-site, and the E-site tRNA exits.

37. What elongation factor drives translocation?

EF-G, which uses GTP hydrolysis to power ribosome movement.

38. Diagram: Ribosome translocation cycle

39. What happens to tRNAs after translocation?

The deacylated tRNA in the E-site is released, and the A-site becomes open for the next aminoacyl-tRNA.

40. What determines when elongation stops?

The appearance of a stop codon (UAG, UAA, UGA) in the A-site.

41. What molecule recognizes stop codons?

A release factor (RF) protein that binds to the A-site.

42. What are the two main actions of release factors?

(1) Hydrolyze the covalent bond between the finished polypeptide and tRNA in the P-site; (2) Trigger disassembly of the ribosome.

43. Diagram: Termination events

44. What happens to ribosomal subunits after termination?

They dissociate into 30S and 50S subunits and are recycled for new translation rounds.

45. Why is bacterial translation considered efficient?

Multiple ribosomes (polysomes) can translate the same mRNA simultaneously, producing many protein copies quickly.

46. What type of energy is consumed during translation?

GTP, used during initiation (IF-2), elongation (EF-Tu, EF-G), and termination (release factor steps).

47. What type of enzyme catalyzes peptide-bond formation?

The 23S rRNA acts as a ribozyme within the 50S subunit.

48. Practice 1 – Identify amino acids: What amino acids do the codons AUG GCU UAA code for?

AUG = fMet, GCU = Ala, UAA = Stop → Polypeptide = fMet-Ala.

49. Practice 2 – Reading frame: If the mRNA is GAUGCUAAG, what is the correct reading frame starting at AUG?

Frame = AUG CUA AG... → codes for fMet–Leu–Stop (if next codon is UAA).

50. Practice 3 – Stop codon nicknames: Match UAG, UAA, UGA to their nicknames.

UAG = amber, UAA = ochre, UGA = opal.