MICR5831 L2: Genetic Machinery Transcription 7/21/25

What RNA is this?

-Carries genetic code instructions for how to create new proteins

mRNA (Messenger)

What RNA is this?

-Decodes mRNA into proteins

tRNA (Transfer)

What RNA is this?

-Subunits of ribosomes (machine that synthesise proteins)

rRNA (Ribosomal)

Which DNA strand is written 5' to 3'?

Coding/sense strand

Which DNA strand is read by RNAPol 3' to 5' from Promoter to Terminator?

Template strand

True or False: mRNA is transcribed and written in the 3' to 5' direction

False, it is 5' to 3'

What does mRNA translation begin with?

Ribosome recognizes RBS (binding site)

How do tRNAs donate amino acids to the growing peptide?

Recognize three-base codon

What Transcription step is this?

-Initiation

RNA polymerase binds promoter and begins transcription at transcription start point

What Transcription step is this?

-Elongation

Successive addition of ribonucleosides to RNA strand

What Transcription step is this?

-Termination

Completed mRNA transcript released

What makes RNA transcription different from DNA replication?

Only one strand is copied during RNA transcription

What does enzymatic RNA synthesis require?

1) Four ribonucleoside 5' triphosphates

2) Magnesium (Mg)

3) DNA template

4) RNAPol

True or False: RNA transcription does not require primers, only a DNA template

True

What cation is required to synthesize RNA?

Magnesium (Mg2+)

What is this?

-Gene

Entire nucleic acid sequence necessary for expression of a gene product

What makes DNA different from RNA?

DNA: Double stranded, 2' carbon has a hydrogen, Thymine

RNA: Single-stranded, 2' carbon has a hydroxyl, Uracil

What direction will this DNA strand be?

-Coding strand

-NOT the strand being transcribed

5' to 3'

What direction will this DNA strand be?

-Template strand

-Transcription based on this

3' to 5'

What direction will this mRNA strand be?

-Complementary mRNA strand

-Complementary to Template DNA strand

5' to 3'

What four ribonucleotide 5' triphosphates are necessary for RNA synthesis?

5' ATP, GTP, CTP, or UTP

Why is Mg so important?

-It helps a lot of enzymes to work

-Without it, hard to sleep

What is the overall reaction for RNA synthesis?

NMP + XTP -> XMP—NMP + PPi

NMP = Peptide chain that receives new nucleotide

XTP = Triphosphate, first nucleotide added to peptide

Ppi = Pyrophosphate, byproduct that is cleaved off

What sugar is present in RNA that has a hydroxyl (OH) at the 2' carbon?

Ribose

How many alpha, beta and sigma units does the RNA polymerase have?

2 alpha

2 Beta

1 sigma factor

What does the RNA polymerase do?

-Bind to promoter region (DNA)

-Unravel/unzip DNA helix

-Recruit ribonucleotide triphosphates

-Match nucleotides to DNA template (5' to 3') base pairs

Which unit of the RNApol is this?

-Initiation factor

-Binds to specific sequences near -10, -35 box

-Recognizes TATA box

Sigma 70 Factor

Where does the Sigma 70 factor interact with the template strand of DNA?

Promoter

Which unit of the RNApol is this?

-Controls the frequency of initiation of transcription

Alpha

Which unit of the RNApol is this?

-Polymerizes NTPs during pyrophosphate reaction

-Transcribes DNA

Beta1, Beta Prime

What does Sigma factor form when it binds reversibly to RNApol?

RNA polymerase holoenzyme

What does RNA polymerase holoenzyme do after Sigma factor binds to RNApol?

-Selectively binds DNA promoter (-10, -35)

-Unravels, unzips DNA

-Initiates mRNA transcription at tsp (+1)

-Releases Sigma factor to allow elongation

What is this?

-Major sigma factor

-Recognizes TATA box promoter (-10 sequences of TATAAT)

Sigma 70

What are some possible physiological signals that could cause minor sigma factors to be expressed?

-Starvation

-Temperature change

-Growth phase

True or False: Each sigma factor recognizes a different promoter, so RNApol will transcribe different genes based on what physiological signals are present

True

What does it mean for RNA to be self-complementary after RNApol reaches the termination sequence?

-Binds to itself when it exits RNApol

-mRNA forms hairpin loop and is released

-RNA polymerase can dissociate

What formation does mRNA make after the termination sequence, allowing it to be released from RNApol?

Hairpin loop

Where would you find the hairpin loop?

3' UTR (Untranslated region)

True or False: Amino acids can directly recognize the mRNA codons

False, they need tRNAs as adaptors

What is this?

-tRNAs

-Small adaptor molecules

-Align specific amino acids with triplet codons in mRNA during translation

Name some example of bases beides A, C, U, G that could be found in tRNA due to post-transcriptional modification

-Inosine (modified adenine)

-Dihydrouridine

-Pseudoiridine

What creates the cloverleaf structure associated with RNA?

Internal complementary base pairing

What is this?

-3 base sequence

-Determines mRNA codon binding

Anticodon

Where does the amino acid attach to tRNA?

3' end

What are some examples of modified bases in tRNA?

-Dihydrouracil

-Pseudouridine

True or False: The 3D shape of a tRNA determines which amino acid is attached by aminoacyl-tRNA syntheases

True

What is this?

-Couples a particular amino acid to its corresponding tRNA

Aminoacyl-tRNA synthease

What happens during amino acid activation?

1) Aminoacyl-tRNA synthetase couples amino acid to its tRNA

2) tRNA whose anticodon forms base pairs with mRNA codon

Where does the energy for linking amino acid to tRNA come from?

ATP is hydrolyzed to create the high energy bond

True or False: The bond formed between the tRNA and amino acid is a low energy bond

False

True or False: For each amino acid, there are several aminoacyl-tRNA synthetases that all correspond to the same single amino acid

True

What enzymes are responsible for attaching the correct amino acid (as specified by the anticodon) to the tRNA 3' end?

Aminoacyl-tRNA Synthetases

What synthetase attaches leucine to tRNAleu?

What synthetase attaches alanine to tRNAAla?

Leucyl-tRNA

Alanyl-tRNA

What is it called when the correct amino acid is attached to its tRNA?

-Charged/acylated

-tRNA becomes amino acyl tRNA

What is it called when an incorrect amino acid is attached to the tRNA?

Mischarging

True or False: Ribosomes are an integral part of ribosomal structures and important parts of protein synthesis machinery

True

Which rRNA is found in prokaryote Large Ribosomal Subunit (50S)?

5S

23S

Which rRNA is found in prokaryote Small Ribosomal Subunit (30S)?

16S

How is rRNA transcribed?

-Tandem repeats

-Two DNA genes are each transcribed by RNApol

-Terminal ribosomes at ends of rRNA

True or False: During optimal growth, 80% of RNA synthesis can be dedicated to rRNA and tRNA transcription

True

What percent of the genome do rRNA and tRNA genes represent?

1 percent

What are the features of a gene required for transcription? (slide 4)

-Promoter

-Shine Dalgarno sequence

-ORF

-Start and stop codons

-Transcription start and termination points

What is the composition of RNA? (slide 5)

-Ribose sugar

-A, C, G, U

-Single strand

What are the three types of RNA and what are their roles? (Slide 6)

-Messenger RNA: Carries genetic code instructions for how to create new proteins

-Transfer RNA: Decodes mRNA into proteins

-Ribosome RNA: Subunits of ribosomes (machine that synthesize proteins)

What are the conventions for DNA during transcription? (you will be given an unlabeled figure to annotate) (slide 7 and 8)

-DNA sequences are written in the 5' to 3' direction

-Coding/sense DNA is 5'->3'

-Template strand is 3' to 5'

-Transcribed mRNA is 5' to 3'

-mRNA translation starts with RBS recognition

-tRNAs donate amino acids by recognizing three base codon

What are the necessary components for transcription?

Note: NOT features of a gene, actual ingredients

(slide 10)

1) Four ribonucleoside 5' triphosphates (5' ATP, GTP, CTP and UTP)

2) Magnesium (Mg2+)

3) DNA template (no primer required)

4) RNAPol (initiation, elongation, termination)

What is the overall reaction for transcription? (slide 11)

NMP + XTP -> XMP--(NMP) + PPi

XMP and NMP are linked via phosphodiester

XTP: First Nucleotide of the chain

Ppi: Pyrophosphate byproduct

What are the components of RNA polymerase? (slide 12)

-Multi subunit complex

-2 alpha: aI, aII

-2 beta: B, B'

-1 sigma factor

How does RNA polymerase generate RNA from DNA? (slide 12

-Binds to promoter

-Unravels and unzips DNA, recruits ribonucleoside triphosphates

-Matches them to DNA template (5' -> 3') via base pairing

Describe the first four steps in transcription initiation (slide 13)

1) Sigma factor binds reversibly to RNA polymerase to form holoenzyme

2) Selectively bind promoter region (-10 and -35)

3) Unravels/unzips DNA helix

4) Initiates mRNA transcription at tsp (+1)

What are the roles of the sigma factor in transcription initiation (slide 14)

-Forms holoenzyme with RNApol

-Unwinds and unzips DNA helix

-Recognizes TATA box promoter

What causes transcription termination? (slide 15)

-RNApol reaches the termination sequence

-mRNA forms hairpin loop (self-complementary region)

-Newly synthesize mRNA

-RNApol dissociates from DNA

What features of the mRNA are required for translation? (slide 17)

-Shine Dalgarno sequence

-Start and stop codon

-5' and 3' UTR

What is a tRNA? (slide 18)

-Adaptor small molecules

-Align specific amino acids opposite their triplet codon in the mRNA molecule during translation

Draw and label the structure of tRNA. (Slide 19)

-Clover leaf structure

-Anticodon sequence of 3 bases (at bottom) determines mRNA codon binding

-D loop, T loop (connected to acceptor stem)

-3' end at top with acceptor stem, longest end

-5' end bound to acceptor stem/3' end

Describe how the structure of tRNA is formed? (slide 19)

-Internal complementary base pairing of RNA

-Gives clover leaf structure

Name two modified bases found in tRNA (slide 19)

D = dihydrouracil

Ψ = pseudouridine

How are amino acids linked to tRNAs (slide 21)

-Aminoacyl-tRNA synthetase couples a particular amino acid to its corresponding tRNA

-tRNA anticodon forms base pairs with codon of mRNA

What is the composition of a ribosome (slide 23).

Large 50S Subunit: 5S, 23S

Small 30S Subunit: 16S

How is a ribosome formed? (slide 24)

-Transcribed, folded, modified in tandem repeats by RNA polymerase

-Ribosomal proteins bind together, form 30S and 50S subunit

-Increased transcription during optimal growth along with tRNA