Lecture Connections 26 | RNA Metabolism

Overview of RNA Function• Ribonucleic acids play three well-understood roles in living cells – Messenger RNAs encode the amino acid sequences of all the polypeptides found in the cell –

Lecture Connections

26 | RNA Metabolism

© 2009 W H Freeman and Company

Overview of RNA Function

• Ribonucleic acids play three well-understood roles in living cells

– Messenger RNAs encode the amino acid sequences of all the polypeptides found in the cell

– Transfer RNAs match specific amino acids to triplet codons in mRNA during protein synthesis

– Ribosomal RNAs are the constituents and catalytic appropriate amino acid

• Ribonucleic acids play several less-understood

functions in eukaryotic cells

– Micro RNA appears to regulate the expression of genes,

possibly via binding to specific nucleotide sequences

• Ribonucleic acids act as genomic material in viruses

Overview of RNA Metabolism

• Ribonucleic acids are synthesized in cells using DNA as

a template in a process called the transcription

– Transcription is tightly regulated in order to control the

concentration of each protein in the cell at optimal level

• Being mainly single stranded, many RNA molecules can fold into compact structures with specific functions

– Some RNA molecules can act as catalysts (ribozymes), often

using metal ions as cofactors

• Most eukaryotic ribonucleic acids are processed after synthesis

– Elimination of introns; joining of exons

– Poly-adenylation of the 3’ end

– Capping the 5’ end

Transcription in E coli

• The nucleoside triphosphates add to the the 3’ end of the growing RNA strand

• The growing chain is complementary to the

template strand in DNA

• The synthesis is catalyzed by enzyme ( RNA polymerase )

• RNA polymerase covers about 35 bp-long

segment of DNA

Replication vs Transcription

• Both add nucleotides via an attack of the 3’ hydroxyl of the growing chain to -phosphorus of nucleoside triphosphates– RNA synthesis requires ribonucleoside triphosphates

– RNA synthesis pairs A with U instead of dA with dT

• Both require catalysis by a Mg++-dependent enzyme

– RNA synthesis has lower fidelity

– RNA synthesis does not require a primer for initiation

• Both require a single strand of DNA as molecular template for building the new strand

Both DNA Strands may Encode for

Proteins

• Adenovirus is one of the causative agents of

common cold

• Adenovirus has a linear genome

• Each strand encodes for a number of proteins

RNA Synthesis is Catalyzed by the

RNA Polymerase

• Mg++ on the right coordinates to the  -phosphate and stabilizes the negatively charged transition

state

Movement of RNA Polymerase

Causes Local Supercoiling

• Positive supercoils (overwound) ahead of the bubble

• Negative supercoils (underwound) behind the bubble

• Topoisomerase eliminates positive supercoils

Bacterial RNA Polymerase has at

Least Six Subunits

• Two two  subunits function in assembly and

binding to UP elements

• The  subunit is the main catalytic subunit

• The  ’ subunit is responsible for DNA-binding

• The  subunit directs enzyme to the promoter

• The  appears to protect the polymerase from denaturation

The  Subunit Binds to Promoter

 Subunit Determines the Types

of Genes Expressed

Transcription Initiation and

 -Independent Termination of

Transcription in Prokaryotes

• The RNA polymerase pauses at certain

sequences during transcription

• Some sequences allow formation of the hairpin within the product

• If the polymerase pauses for long enough and the hairpin forms, the RNA-DNA hybrid is

disrupted

• This promotes dissociation of the polymerase

Eukaryotes Contain Several

Distinct Polymerases

• RNA polymerase I synthesizes pre-ribosomal RNA

(precursor for 28S, 18S, and 5.8 rRNAs)

• RNA polymerase II is responsible for synthesis of mRNA

– Very fast (500 – 1000 nucleotides / sec)

– Specifically inhibited by mushroom toxin -amanitin

• RNA polymerase III makes tRNAs and some small RNA products

• Plants appear to have RNA polymerase IV that is

responsible for the synthesis of small interfering RNAs

• Mitochondria have their own RNA polymerase

Eukaryotic mRNA Transcription

Involves Several Proteins

Assembly of RNA Polymerase

• Assembly is initiated by interaction of binding protein with the promoter

TATA-• Helicase activity in TFIIH unwinds DNA at the promoter

• Kinase activity in TFIIH phosphorylates the polymerase allowing the latter to escape the promoter

DNA Recognition with

TATA-Binding Protein is Well Understood

Architecture of the Elongation

Complex

Formation of Primary Transcript and Its Processing in Eukaryotes

• The removal of introns from the primary transcript

is called splicing

RNA Processing

• Almost all newly synthesized RNA molecules (primary transcripts) are processed to some degree in eukaryotic cells

– The 5’-end is capped w/ methylguanosine

– Introns are spliced out

– Poly-A tail is built at the 3’ end

• Processing is catalyzed by protein-based enzymes and

by RNA-based enzymes (ribozymes)

• Only some prokaryotes have to splice out introns but many process their tRNA precursors

Capping the 5’ of mRNA

• Capping protects mRNA from 5’exonuclease degradation

Capping Enzymes Are Tethered to the C-terminal Domain of Polymerase II

Four Major Groups of Introns

• Spliceosomal introns are spliced by splicesomes

– These are most common introns

– Frequent in protein-coding regions of eukaryotic genomes

• Group I and Group II introns are self splicing

– Interrupt mRNA, tRNA and rRNA genes

– Found within nuclear, mitochondrial, and chloroplast genomes – Common in fungi, algae, and plants, also found in bacteria – Group I and Group II differ mainly by the splicing mechanism

• tRNA introns are spliced by protein-based enzymes

– Found in certain tRNAs in eukaryotes and archae

– Primary transcript cleaved by endonuclease

– Exons are joined by ATP-dependent ligase

Splicing of Group I Introns

Transesterification in Splicing of Group I and Group II Introns

• In case of group I intron splicing, the

nucleophile is 3’ hydroxyl of free guanosine, GMP, GDP, or GTP

• In case of group II intron splicing, the

nucleophile is an hydroxyl in RNA

Splicing of Group II Introns

Overview of RNA Processing

Chapter 26: Summary

• RNA polymerase synthesizes RNA using a strand of DNA as

a template and nucleoside triphosphates as substrates

• The primary RNA transcript in eukaryotes requres processing before it becomes messanger RNA

• The processing involves capping 5’end with methylguanosine

to stabilize the RNA molecule

• The processing involves splicing out introns

• Some introns have an amazing ability to carry out their own splicing

In this chapter, we learned that: