Lecture 3 - Transcription and Translation

What is the process whereby genes make pre-mRNA?

Transcription

What happens during transcription in eurkaryotes?

makes mRNA

What happens during transcription in prokaryotes?

makes RNA (no modification)

What additional steps occur in eukaryotes between transcription and translation?

Post-transcriptional RNA processing.

Does post-transcriptional RNA processing occur in prokaryotes?

No, processing is not required

What are the major steps of gene expression in eukaryotes?

Transcription → Post-transcriptional RNA processing → Translation → Post-translational processing.

What happens during post-translational processing?

Folding, chemical modification, localization, cleavage, and/or assembly with other proteins.

What is transcription?

Production of RNA from a DNA template.

Can DNA in eukaryotic organisms leave the nucleus?

No it must stay to be copied.

During transcription, how many DNA strands are copied?

One strand.

During replication, how many DNA strands are copied?

both strands are copied.

What is the template strand?

DNA strand used to synthesize RNA.

What enzyme is involved with transcription?

RNA Polymerase

What is the non-template strand also called?

Coding (sense) strand.

How does RNA sequence compare to the coding strand?

Same sequence except U replaces T.

Why does RNA need to be processed in the nucleus?

For protection from cytoplasmic environment.

Why do we need to protect the RNA?

So it can be translated.

What is the production of protein from RNA?

Translation

Why are proteins critical for transcription/translation?

they act as the essential enzymes, motors, and regulators that catalyze, facilitate, and control every step of gene expression

What directional does RNA polymerase run?

5’ to 3’ direction.

What is the template strand also called?

Anti sense strand

What are the three parts of a transcription unit?

Promoter, RNA-coding sequence (gene), terminator.

What is the purpose of the promoter?

Polymerase binding site

What is the transcriptional start site?

Where the RNA Polymerase starts making RNA.

What is the transcriptional termination site?

Where polymerase falls off.

What does the RNA transcript include?

It includes everything after the transcription start site and stops after the transcription terminal site.

Is the promoter copied into RNA?

NO

Is the terminator usually copied?

Yes.

What is RNA-coding region?

The portion of an mRNA transcript that is translated into protein

Three phases of bacterial transcription?

Initiation, elongation, termination.

What happens during initiation?

Transcription machinery assembles at promoter.

What happens during elongation?

RNA polymerase synthesizes RNA.

What happens during termination?

RNA released from DNA.

What are consensus sequences?

Promoter sequences recognized by transcription machinery.

Why is the 3’ OH group always near polyermase?

Polymerase needs 3’ OH group to build.

What happens directly after transcription in Prokaryotic organisms?

Ribosomes attaches and starts translating.

What is Rho?

Protein that is able to bind to mRNA

What is Rho utilization sequence (rut)?

A sequence that is found on the RNA molecule that is capable of binding the Rho protein.

What happens after Rho protein binds to the Rho utilization sequence?

It attaches to the mRNA and crawls up the RNA, moving toward the polymerase. It then budges the polymerase off the DNA (forcing termination).

What is rho-dependent termination?

Rho binds rut site on RNA and unwinds RNA-DNA hybrid.

What is rho-independent termination?

Hairpin secondary structure destabilizes RNA-DNA hybrid.

How does a secondary sequence form?

hairpin due to Complimentary within the structure

What does the secondary structure do?

It pulls the RNA away from the RNA polymerase, destabilizing the entire structure.

How is eukaryotic transcription different from bacterial?

Requires more proteins and complex promoters.

What are enhancers?

Sequences that are found on the DNA that are capable of binding activors.

What do activator proteins do?

Stabilize all of the mediator proteins that keep polymerase attached.

What are nucleosomes?

DNA wrapped in histones

How do nucleosomes affect transcription?

Histones block transcription factor binding.

What loosens histone-DNA interactions?

Acetylation of lysines.

Why does acetylation loosen DNA?

Adds negative charge, reducing attraction to DNA.

What does histone acetyltransferase (HAT) do?

It is an enzyme that transfers free floating acetyl groups and attach them to the tail of histone

What does deacetylation do?

 it removes the acetyl group so that the lysine are positively charged and can grip the DNA tightly. 

What does RNA polymerase I transcite?

Large rRNA

What does RNA polymerase III transcribe?

tRNAs

What does RNA polymerase II transcribe?

mRNA —> protein

How many proteins assemble with RNA pol II at promoters?

~50 polypeptides.

How many nucleotides synthesized before elongation phase?

30 base pairs (bp)

What happens to initiation factors when high expression is needed?

Remain on promoter.

How does Pol I terminate?

Protein binds downstream DNA sequence.

How does Pol II terminate?

Rat1/Xrn1 degrades RNA.

What is different during Pol II termination?

Poly II will continue to work past the termination sequence. Enzymatic reaction (cutting mechanism) releases the transcript. Rat1/Xrn1 binds to RNA and eats away at the sequence until it gets to polymerase (eats that too).

How does Pol III (tRNA) terminate?

Long stretch of uracils.

What does colinear mean?

Nucleotide number corresponds directly to amino acid number.

Are prokaryotic genes colinear?

Yes.

Why are eukaryotic genes more complex?

Contain introns and exons (which have to be removed during processing)

What contains Intron and Exon?

Pre-mRNA

What are exons?

Coding sequences.

What do exons code for?

Protein

What are introns?

Non-coding sequences.

Relationship between intron size and complexity?

More complex organisms → larger introns.

What is removed during RNA processing?

Introns are removed

What stabilizes ribosome binding in prokaryotes?

Shine-Dalgarno sequence.

Why do RNA structures have untranslated Regions (UTRs)?

regulate gene expression, mRNA stability, transport, and translation efficiency.

Is Shine-Dalgarno translated?

No.

Do prokaryotes heavily modify RNA and why not?

No, Transcription and translation occur simultaneously.

What is added to 5′ end of eukaryotic mRNA?

Methyl-guanine cap.

What type of linkage connects cap?

5′–5′ triphosphate.

What is Polyadenylation?

the enzymatic addition of a poly(A) tail (a sequence of adenine nucleotides) to the 3' end of pre-mRNA

What is added to 3′ end of mRNA?

Poly-A tail.

What is the typical length of a Poly-A tail?

5–250 adenines.

What is the function of poly-A tail?

Stabilizes RNA.

What removes introns?

Spliceosome.

What guides spliceosome?

Consensus sequences.

What sequence feature is essential for intron removal during eukaryotic pre-mRNA splicing?

Conserved GU at the 5′ donor site and AG at the 3′ acceptor site within the intron.

What is alternative splicing?

One gene produces multiple proteins.

Why is alternative splicing important?

Tissue-specific protein expression.

What initiates pre-mRNA splicing?

The 2′-OH of the branch-point adenosine attacking the 5′ splice site.

What unusual bond forms during lariat formation?

A 2′–5′ phosphodiester bond.

What happens in step 2 of splicing?

The 3′-OH of exon 1 attacks the 3′ splice site, ligating the exons.

What binds to mRNA to direct editing?

Guide RNAs.

What changes can occur during RNA editing?

Insertions, deletions, substitutions.

Result of RNA editing?

Protein sequence differs from DNA prediction.

How can you create alternative splicing patterns?

During the process of removing introns you can move different exons out.
ex) thyroid cell creating calcitonin (4 exons linked together)

How is Calcitonin-gene-related peptide (CGRP) made?

Producing an mRNA that contains exons 1,2,3,4,5,6.