Transcription and Translation

What is central dogma?

DNA → mRNA → Protein

What is transcription?

DNA to mRNA

mRNA is synthesized by the…?

Template strand

What is the purpose of the coding strand of DNA?

Contains sequence that is equivalent to encoded mRNA (read 5’ → 3’)

What are the similarities between transcription and DNA synthesis?

Complementary base pairing with template, elongation from 5’ → 3’, catalyzed by polymerase (RNA vs DNA pol)

What are the differences in substrates between transcription and DNA synthesis?

Ribonucleotides utilized instead of deoxyribonucleotides; uridine rather than thymidine nucleotides pair with adenosine

What are the differences in process between transcription and DNA synthesis?

Chains start de novo (no need for primer), one DNA strand in each gene serves as template for transcription, and parental double helix reforms

What does RNA polymerase I produce?

rRNA

What does RNA polymerase II produce?

mRNA and various non-coding RNAs

What does RNA polymerase III produce?

tRNAs, 5S rRNA, and various non-coding RNAs

What are transcription promoters?

DNA sequence that specifies where transcription starts

What does RNA polymerase II require in order to initiate transcription?

General transcription factors to position at promoter, and subsequent release of general transcription factors to enter elongation phase

What is the function of transcriptional activators?

Promote assembly on RNA polymerase II with general transcription factors at promoter; transcriptional activator proteins bind to enhancer site on DNA upstream of promoter

What are the 3 steps of pre-mRNA processing?

5’ capping, splicing, and polyadenylation

What is the capping of 5’ end of pre mRNA do?

Linkage of 7-methylguanosine that stabilizes mRNA, aids in nuclear export, and translation initiation

What occurs during RNA splicing?

Removal of introns and ligation of exons; spliceosome mediated

What is the 5’ splice site called?

Splice donor (GU)

What is the 3’ splice site called?

Splice acceptor (AG)

Once splicing is complete, what occurs to pre mRNA?

New proteins bind to exon-exon junction and form exon-junction complex (EJC)?

What is the function of the exon-junction complex (ECJ)?

Aids in RNA export and nonsense mediated decay

What is exon skipping?

5’ splice site (donor) mutation; deletion of amino acids, can cause possible frameshift that can cause altered reading frame with premature stop codons

What is cryptic splice site?

3’ splice site (acceptor) mutation; intron sequences included in spliced mRNA, reading frame containing intron sequences often contains premature stop codons

What is the processing of the 3’ end of pre-mRNA?

Cleavage proteins recognize sequence on 3’ end and cleave, poly(A) polymerase causes polyadenylation, and the poly(A) tail is bound by poly(A)-binding protein

What is the function of the poly(A) tail and binding proteins?

Stabilize mRNA, aid in nuclear export, and increase translation efficiency

What is the goal of translation?

Synthesize a polypeptide coded by the mRNA using the translation machinery

What is the function of aminoacyl-tRNA-synthetases?

Links a specific amino acid to appropriate tRNA

What are 3 base pairs on a DNA strand called?

Triplet

What are 3 base pairs on a mRNA strand called?

Codon

What are 3 base pairs on a tRNA called?

Anticodon

What are the stop codons?

UAA, UAG, UGA

What is the start codon?

AUG (Met)

What is a missense mutation?

Switching of a base pair can cause different amino acid to be entered into protein

What is a nonsense mutation?

Premature stop codon

What is a silent mutation?

Changing of base pair does not affect amino acid brought by tRNA

What is a frameshift mutation?

Insertion or deletion of base pair affects all downstream codons

Where is the amino acid covalently bonded on a tRNA?

3’ acceptor stem

How are amino acids attached to tRNA molecules?

Carboxyl group of amino acid is linked to 3’OH of tRNA by high-energy ester bond

Accuracy of protein synthesis is dependent on accuracy of…?

Aminoacyl-tRNA synthetases

What is the composition of ribosomes?

Large and small subunits; rRNA and proteins

What is the target of bacterial toxins?

Ribosomes

What are the steps of translation?

Initiation, elongation, and termination

What are the steps of elongation in translation?

1) P site contains tRNA attached to growing polypeptide (peptidyl-RNA), A site binds incoming aminoacyl-RNA

2) Peptide bond formation by peptidyl transferase of ribosome

3-4) Translocation: tRNAs shifted while ribosome advances

What are the three sites of a ribosome?

E, P, A

What are elongation factors?

GTP-binding proteins (GTPases) that aid in translation elongation

When are GTPases active?

When GTP-bound

When are GTPases not active?

When hydrolyzed to GDP

What is the function of EF-1?

Promotes entry of correct aminoacyl-tRNA into A site; basically brings tRNA into A site, then is hydrolyzed to GDP and leaves

What is the function of EF-2?

Promotes ribosome translocation following GTP hydrolysis; basically tells the ribosome to shift down the mRNA

What are the steps of translation initiation?

1) Methionine-linked initiator tRNA loaded onto small ribosome P site, aided by eIF-2 (GTP bound); eIF-2 brings MET tRNA to P site of small subunit

2) 5’ G-cap of mRNA binds to small subunit site

3) Scan for AUG start codon

4) eIF-2 GTP hydrolysis leads to dissociation of initiation factors

5) Large subunit assembles over small subunit with mRNA (sandwich-like)

6) Elongation begins

What occurs during translation termination?

1) Stop codons are not recognized by tRNA

2) Release factors (GTP-bound) bind to A site

3) Addition of H2O to peptidyl-tRNA releases poly-peptide chain

4) Ribosome dissociates

What produces Shiga toxin?

Shigella dysenteriae and Enterohemorrhagic E. coli through food poisoning

What does Shiga toxin target?

Acts on vascular endothelium by breaking it down; inhibits protein synthesis by inactivating ribosome. RNA N-glycoside that removes specific adenine base in critical conserved region of 28 S rRNA

What produces Diphtheria toxin?

Bacterial (corynebacterium diphtheriae) URI (gray membrane covering tonsils)

What does Diphtheria toxin target?

Catalyzes ADP-ribosylation of EF-2 (on a modified histidine residue called diphthamide); inhibits EF-2 and impairs ribosome translocation