Molecular Genetics Final Exam

What amino acid is best for alpha helices?

alanine

What amino acid is the worst for alpha helices?

proline

What does mRNA do

codes for the protein starting with the start codon and ending with the stop codon

What does tRNA do

brings the amino acid and recognizes specific codons

What do ribosome subunits do

binds the mRNA

What happens if there is a mutation in the first or second codon

changes the amino acid, but doesn’t change the chemical nature

What do ribosomes do

build proteins by catalyzing peptide bond formation

What are the 3 sites involved in translation

A, P, E

What happens in the A site in translation

charged tRNA is loaded

What happens in the P site in translation

protein is formed

What happens in the E site in translation

uncharged tRNA is released

What is covalently bound to the 3’ end of a tRNA

amino acid

What are the 8 steps to making a tRNA charged?

1. Introns are spliced out 

2. 5’ leader sequence cleaved by RNAseP 

3. 3’ trailer sequence cleaved by RNAseZ 

4. CCA is added to the tail of the tRNA 

5. tRNA exported out of the nucleus into the cytoplasm 

6. ATP binds amino acid to tRNA synthase 

7. tRNA loads onto tRNA synthase 

8. Anticodon recognizes tRNA

What is the wobble model?

When tRNA binds to more than one codon

In small amino acids, how does tRNA synthase work for kinetic proofreading

tRNA synthase has a pocket that only fits the small amino acid

In large amino acids, how does tRNA synthase work for kinetic proofreading

tRNA synthase has a large pocket that can fit small and large amino acids, second pocket that only fits small amino acid and cleaves it

What are the 5 steps for translation elongation

1. tRNA is in the P site at the start codon

2. tRNA is loaded into the A site 

3. Peptide bond forms between amino acid in P site and amino acid in A site 

4. tRNA’s shift 1 site upstream 

5. E site loads

Help the ribosome move alone or position tRNAs

elongation factors

What does EF-Tu do

binds GTP and loads/positions the incoming tRNA

What are the 6 steps for EF-Tu to bind

1. tRNA binds anticodon 

2. EF-Tu spends GTP 

3. GTP converted to GDP 

4. tRNA is moved into the A site 

5. Tu binds to Ts 

6. GTP cleaves Ts and reloads Tu

What does EF-G do

binds GTP and moves everything down

mRNA is translated in a ____________ way

non-overlapping

What are the 6 steps for bacterial translation initiation

1. RBS has a Shine Dalgarno Sequence upstream of AUG

2. Scans along 30S subunit to find the AUG

3. 30S recruited to shine dalgarno 

4. Proteins stabilize sequence fMet-tRNA is recruited to AUG site

5. Large subunit is recruited

6. Bacterial operons express large mRNAs with multiple ORFs with different polycistrons

What are the 8 steps for eukaryotic translation initiation

1. Ternary complex forms 

2. 43S pre-initiation complex forms in the cytoplasm

3. 5’ cap binding proteins recruit 43S to 5’ end of mRNA 

4. 43S scans along the RNA using ATP 

5. 43S reaches AUG 

6. 43s stalls at AUG 

7. 48S initiation complex forms

8. 60S forms with the 48S into the 80S complex

What is the function of the kozak sequence

determines the AUG initiation

What happens when there is a strong kozak sequence

 ribosome stalls and begins translation

What happens when there is a weak kozak sequence

AUG is skipped, translation does not start

genes that only produce 1 protein

monocistronic

What is an example of a monocistronic gene

mRNA

What does the release factor do in translation

cleaves the peptide chain off of the tRNA in the P site

What are the 8 steps to translation termination

1. RF1 binds 

2. Polypeptide released 

3. RF3 dissociates 

4. RRF binds to the stop codon 

5. RRF interacts with EFG 

6. tRNA released in the E site 

7. EFG does GDP hydrolysis 

8. IF3 causes the ribosome to dissociate

What 3 components are required for translation

mRNA, tRNA, ribosome subunits

What is the component that actually carries out translation

ribosome

Give an example of a:

Enzyme

DNA Binding protein

Allosteric interaction

Active transport

Enzyme - Protease

DNA binding protein - Gal4

Allosteric interaction - exposure of protease site and cleavage

Active transport - Gal4 entering the nucleus

What type(s) of polymerase synthesize 3’ to 5’

None

Why is it okay that RNA polymerase has worse fidelity

the errors produced during transcription not inherited by offspring

In eukaryotes, how are the ends of chromosomes differentiated from double stranded breaks

Chromosome ends are protected by telomeres

What does DNA ligase do in DNA replication

seals breaks in the backbone by forming phosphodiester bonds

What does gyrase do in DNA replication

relieves supercoiling ahead of the replication fork by introducing negative supercoils

Which eukaryotic RNA polymerase produces the most RNA in the cell

RNA polymerase 1

How does RNA polymerase synthesis occur?

5’ to 3’

What does RNA polymerase 2 do?

Transcribes mRNA

What does RNA polymerase 1 do?

Encodes small RNAs that don’t need caps and are not meant to be translated (rRNA and micro RNA)

What does RNA polymerase 3 do?

Encodes for small RNAs and histones

What is an operon?

a series of genes that function together

What does RNA polymerase need to function?

DNA template, rNTPs, Mg²⁺/Mn²⁺, promoter, transcription factors

What are the 5 steps of transcription initiation through RNA polymerase

1. Polymerase and sigma factors bind promoter

2. DNA unwinds

3. Open complex forms

4. First rNTP pairs with template

5. Phosphodiester bond forms

How does RNA elongate

Polymerase moves 5’→3’, adds rNTPs complementary to template, DNA rewinds behind

How does transcription stop in bacteria

rho-dependent or rho-independent

How does transcription stop in eukaryotes

polyadenylation signal → RNA cleaved and released

what nucleotides does RNA polymerase use

ATP, GTP, CTP, UTP

How do mistakes occur by RNA polymerase

Wrong rNTP pairs with DNA template because RNA polymerase has no strong proofreading like DNA polymerase

How does RNA polymerase fix mistakes (2 ways)

• Backtracking: Polymerase moves backward on the RNA.

• Cleavage of RNA: The misincorporated nucleotide is cut out.

How are transcripts modified by RNA polymerase?

5’ capping, 3’ poly-a tail, splicing

Why is a 5’ cap important?

RNA stability, translation, and transporting RNA out of the nucleus

Why is a 3’ poly-a tail important

mRNA stability

What does RNA splicing do?

Determines which codon is being used

4 steps to operon function

1. RNA pol binds promoter

2. Repressor binds operator

3. Activator binds DNA

4. Structural genes code proteins

In the lac operon, regulation of _____ is done by the control of ____________

Initiation, RNA polymerase binding

In the trp operon, regulation of ________ is done by _________

elongation/termination, attenuation

What does attenuation cause

premature termination of transcription under certain conditions

a sequence consisting of two identical or highly similar inverted repeats which are either adjacent to one another or separated by a spacer region

Palindrome

What is an example of a palindrome in bacteria

Transcription factor binding sites

In the lac operon, what level of gene expression is present when there is low glucose, high cAMP, no lactose

None

In the lac operon, what level of gene expression is present when there is : high glucose, low cAMP, present lactose

Low

In the lac operon, what level of gene expression is present when there is low glucose, high cAMP, present lactose

High

How is bacterial initiation regulated?

Sigma factors, activators, and repressors

How do repressors work in bacteria?

Bind to the operator and prevent RNA polymerase from initiating transcription

What inactivates repressors in bacteria

Inducers

How do activators work in bacteria?

Bind to DNA near the promoter and help RNA polymerase bind

What do activators in bacteria need to function

Co-activator or ligand

What is the default state of a promoter in bacteria?

On

How is eukaryotic initiation regulated? (5 things)

• Promoter

• Activators/Repressors

• Mediator

• Chromatin

• Coactivators/Corepressors

What is the promoter in eukaryotic transcription and what does it do

TATA box ; recruits RNA Pol II

What do activators/repressors do in eukaryotic transcription

Bind enhancers/silencers to boost or block transcription

What does the mediator do in eukaryotic transcription

Bridges TFs to RNA Pol II and integrates signals

What does chromatin do in eukaryotic transcription

Histone modifications and nucleosome remodeling control accessibility

What do coactivators/corepressors do in eukaryotic transcription

Modify chromatin or stabilize polymerase

What do activators do in eukaryotes

Bind enhancers to help RNA Pol II initiate transcription

What do repressors do in eukaryotes

Bind silencers to block transcription

What is the default state of a promoter in eukaryotes

Off

How is the beginning of the transcript modified?

5’ capping

Describe Rho-independent transcript termination in bacteria (3 steps)

1. RNA forms a hairpin loop and poly-U sequence

2. Hairpin destabilizes RNA-DNA hybrid

3. Polymerase releases RNA

Describe Rho-dependent transcript termination in bacteria (5 steps)

1. Rho protein binds rut site on RNA

2. Moves along RNA in the 3’ direction

3. RNA polymerase reaches the termination site

4. Stem-loop causes RNA polymerase to pause

5. Helicase separates RNA from DNA

How is the end of the transcript modified (5 steps)

1. polyA signal recognized

2. RNA cut downstream of signal

3. PolyA polymerase adds A’s

4.  Poly-A binding proteins bind tail

5. Rat1/hXrn2 degrades leftover RNA

Short single-stranded DNA or RNA that bind to specific targets with high affinity and specificity

Aptamers

Aptamer tied to a regulatory structure; Regulate gene expression when there are environmental or cellular changes

Riboswitches

Regulatory region of 100,000 base pairs upstream of the promoter; Lots of space that influences if the region is on or off

INK/ARF locus

bind activators and transcription factors to start transcription

Enhancers

How does splicing occur mechanistically? (7 steps)

1. 2’OH attacks 5’ splice site

2. Exon 1 released

3. 2’OH forms covalent bond with 5’ end of exon

4.  3’OH attacks 3’ splice site

5. Exon 1 and exon 2 link

6. Intron lariat released

7. Spliced RNA processed

Why does splicing occur? (3 reasons)

1. Removes non-coding introns

2. Joins exons together to produce a continuous open reading frame

3. Allows alternative splicing

What is alternative splicing?

A process where different combinations of exons are joined from a single pre-mRNA

Describe the 2 types of alternative splicing

1. Minor spliceosome - recognizes AU5’ and AC 3’

2. Self-splicing introns - introns that splice themselves with only RNA, no proteins involved

How does alternative splicing affect transcripts?

allows a single pre-mRNA to produce multiple mature mRNA isoforms

removed pieces of RNA

intron

kept and translated during RNA processing

exon

large complex that does the splicing

spliceosome

splice donor

5’ splice site

splice acceptor

3’ splice site

proteins coded from the same gene but are spliced differently

isoforms