26| RNA Metabolism

© 2013 W. H. Freeman and Company

CHAPTER 26 RNA Metabolism

– Transcription: DNA-dependent synthesis of RNA– Capping and splicing: RNA processing

Key topics:

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 acids

• Ribonucleic acids play several less-understood functions in eukaryotic cells:

– MicroRNA 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 transcription– Transcription is tightly regulated in order to control the concentration

of each protein

• 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 a 35 bp-long segment of DNA

Transcription by RNA Polymerase in E. coli

Features of Transcription

• RNA polymerase binds to sequence called promoter to begin transcription– Primer not required

• Growing end of new RNA temporarily base-pairs with DNA template for ~8 bp

• DNA duplex unwinds, forming a “bubble” of ~17 bp

• RNA Pol generates positive supercoils ahead, later relieved by topoisomerases

Transcription “Bubble”

RNA Polymerase and Generation of Positive Supercoils

Be careful of terminology

• DNA Template Strand – serves as template for RNA polymerase

• DNA Coding Strand – the non-template strand; has the same sequence as the RNA transcript

– See Fig. 26-2

***Regulatory sequences are listed by the coding strand sequence.

Template and Coding Strands

Both DNA strands may encode for proteins

• Coding information may be on either strand (“top” or “bottom”) • See Fig. 26-3 for organization of coding information in the adenovirus genome• Adenovirus is one of the causative agents of the common cold• Adenovirus has a linear genome• Each strand codes for a number of proteins

Coding Organization in an Adenovirus Genome

RNA polymerase is a large enzyme with no proofreading capability

• RNA polymerase holoenzyme has five core subunits of 2’ plus a sixth called

• See Fig. 26-4

• RNA Pol lacks 3’ 5’-exonuclease, so has high error rate of 1/104–1/105

• RNA binds to promoter regions to initiate transcription

Bacterial RNA polymerase has at least six subunits

• Two subunits function in assembly and binding to UP (upstream promoter) elements– See slide on promoters and Fig. 26-5

• 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

Promoters in E. Coli that bind the same RNA polymerase have common features

• Two consensus sequences at −10 (TATAAT) and −35 (TTGACA) for subunit binding– Called TATA sequences

• A-T−rich upstream promoter element between −40 and −60 binds the subunit

• A-T−rich sequences promote strand separation• These sequences govern efficacy of RNA Pol

binding and therefore affect gene expression level

Some E. coli Promoters

The footprinting technique is a way to find a DNA-binding site

Premise: DNA bound by protein will be protected from chemical cleavage at its binding site.

1)Isolate a DNA fragment thought to contain a binding site2)Radiolabel the DNA3)Bind protein to DNA in one tube; keep another as a “naked DNA” control4)Treat both samples with chemical or enzymatic agent to cleave the DNA5)Separate the fragments by gel electrophoresis and visualize bands on X-ray film or imager plate

Protein-DNA Footprinting

Footprinting Results of RNA Polymerase Bound to Promoter

Transcription initiation and elongation have several steps

• See Fig. 26-6• RNA Pol binds to promoter

– Creates a closed complex (DNA is not unwound)• Open complex forms

– Region from ~−10 to ~+2 unwinds• RNA Pol moves away from promoter

is replaced by protein NusA

Transcription Initiation and Elongation in E. coli

Transcription is a major target for regulation

• Transcription is energy-intensive so it’s logical to regulate gene production here

• Regulation is achieved in many ways – One way is to regulate the affinity of RNA

polymerase for a promoter• Promoter sequence• Activator proteins• Repressor proteins

Two Types of Termination in E. Coli

1) -independent– Characterized by three Us near the 3’ end of

the transcript– Self-complementary regions in transcript form

a hairpin 15−20 nt before the 3’ end• Makes the RNA Pol pause, dissociate

2) -dependent– See next slide

The -dependent pathway is less understood

• Known: common CA-rich sequence called a rut site (Rho utilization element)

protein processes until termination site reached

protein is a helicase, binds to rut site• See Fig. 26-7b

Termination of Transcription in E. coli by -Independent Pathway

Termination of Transcription in E. coli by -Dependent Pathway

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– Can recognize thousands of promoters (See Fig. 26-8)

• 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

Features of Some Promoters Recognized by Eukaryotic RNA Polymerase II

Eukaryotic mRNA transcription involves many proteins

• Relies on protein-protein contacts– Many highly conserved transcription factors

• RNA Pol II is well-studied– Large complex of 12 subunits

• Some subunits have some structural homology to bacterial RNA polymerase

– Has carboxy-terminal domain of highly conserved repeats

Transcription at RNA II Promoters

Assembly of RNA Polymerase at Promoter

• Initiated by TATA-binding protein (TBP) with the promoter-TBP is part of multisubunit complex TFIID

• Other proteins include TFIIB, TFIIA, TFIIF, TFIIE and TFIIH

• Helicase activity in TFIIH unwinds DNA at the promoter

• Kinase activity in TFIIH phosphorylates the polymerase at the CTD (carboxy-terminal domain) changing the conformation and enabling RNA Pol II to transcribe

Elongation and Termination

• Elongation factors bound to RNA Pol II enhance processivity and coordinate post-translational modifications

• For termination, Pol II is dephosphorylated• Regulation is complex (see Chapter 28)

TFIIH and Repair

• Transcribed genes are more actively repaired than silent genes

• May partly be explained that TFIIH also has role in nucleotide-excision repair (NER)– Recruits the NER complex at a lesion

• Genetic repair diseases are associated with TFIIH defects– Xeroderma pigmentosum, etc.

RNA polymerases can be selectively inhibited

• Actinomycin D and Acridine– Intercalate in DNA and prevents transcription

• Rifampicin– Binds to -subunit of bacterial RNA Pols

-Amanitin from mushroom Amanita phalloides– Blocks Pol II and Pol III of predators– But doesn’t block its own Pol II

Processing of mRNA − Overview

• Dozens of proteins coordinate with each other and with proteins involved in RNA transport to ribosomes

• Processing includes:– Splicing out introns and rejoining any exons for a

continuous sequence– Adding a 5’-cap– Adding a 3’-poly(A) tail– Degradation

Maturation of mRNA in Eukaryotes

The 5’-cap is a 7-methylguanosine

• 7-methylguanosine links to 5’-end via 5/,5’-triphosphate link– May include additional methylations at 2’OH

groups of next two nucleotides– Methyl groups derive from S-adenosylmethionine

(SAMe)• See Fig. 26-12• Protects RNA from nucleases• Forms a binding site for ribosome

The 5’-cap of mRNA

Introns are found in most genes

• Most genes in vertebrates, some in yeast, a few bacteria have introns

• Exons usually <1000 bp in length• Introns 50−20,000 bp in length• Some genes have dozens of introns

Four Classes of Introns

• Group I and Group II introns are self-splicing– Require no additional proteins or ATP– In nuclear, mitochondrial, and chloroplast genomes– Differ mainly in the splicing mechanism

• Spliceosomal introns are spliced by enormous complexes called splicesomes– The most common introns– Frequent in protein-coding regions of eukaryotic genomes

• tRNA introns are spliced by protein-based enzymes– Primary transcript cleaved by endonuclease– Exons are joined by ATP-dependent ligase

Splicing of Group I Introns

• See Figs. 26-13 and 26-14• 3’-OH of free guanosine (GMP, GDP, etc.) is

used as a nucleophile• Attacks phosphodiester bond between U

and A at the end of the intron• Releases first portion of U-ending exon• The 3’-OH of the U-ending exon then

attacks the 5’-end of the other piece of exon to rejoin the pieces

Nucleophilic Attack of Guanoisine 3’-OH on UA of Exon-Intron Interface in Group I Intron

Splicing Mechanism of Group I Intron

Group II introns use a 2’-OH within the intron as a nucleophile

• The nucleophile is a 2’-OH of an A residue within the intron

• After the first cleavage, the second (right-most) piece forms a lariat-like intermediate with a 2’-5’-phosphodiester bond

• Other features are similar to Group I introns• See Fig. 26-15

Splicing of Group II Intron

Spliceosome introns are removed via a large complex called a spliceosome

• Spliceosome made up of snRNPs (“snurps” for small nuclear ribonuclear proteins)– snRNP RNA is called snRNA (for small nuclear

RNA)• 5 snRNAs known in eukaryotes (U1, U2, U4, U5, U6)

• GU at 5’-end and AU at 3’-end usually mark sites of splicing

U1 snRNP and U2 snRNP bind to the intron’s ends

• Contain regions complementary to mRNA• U1 helps define the 5’-splice site• U2 binds near the 3’-end of the intron

– Creates a bulge that partly displaces and activates an A to be a better nucleophile

– This A forms the 2’5’-phosphodiester bond of the lariat-like intermediate

• See Fig. 26-16a

Binding of U1 and U2 snRNP to mRNA

• Next, U2, U4, U5, and U6 bind, bringing at least 50 proteins to create spliceosome

• ATP required for assembly but not cleavage• Some parts attached to CTD (carboxy-

terminal domain) of RNA Pol II– Indicates coordination of splicing with

transcription

U1 snRNP and U2 snRNP bind to the intron’s ends (cont.)

Poly(A) tail is added to eukaryotic mRNAs to serve as a binding site

• RNA Pol II synthesizes RNA beyond the cleavage signal sequence– Cleavage signal is bound by an endonuclease and a

polyadenylate polymerase bound to CTD• Endonuclease cleaves RNA 10−30 nt downstream

to highly conserved AAUAA• Polyadenylate polymerase synthesizes 80−250 nt

of A• See Fig. 26-17

Addition of Poly(A) Tail

Overview of mRNA Processing

A single gene can yield different products depending on RNA processing

• RNA can be “edited” (bases removed/added)• Cleavage/polyadenylation patterns can vary,

yielding different mature transcripts• Immunoglobulin heavy chain gene: different

degrees of polyadenylation and different cleavage sites yield diverse sequences

• Calcitonin and calcitonin-gene-related peptide, in rat thyroid and brain, respectively, made from same mRNA

Alternative Splicing Mechanisms

Alternative Splicing in the Calcitonin Gene of Rats

Processing of tRNA and rRNA

• Bases are modified in post-transcription rxs (see Fig. 26-22)– Pseudouridine ()– Thiouridine– Dihydrouridine, etc.

• rRNAs and tRNAs are cleaved from longer precursors

Modified Bases in tRNAs and rRNAs

MicroRNAs Function in Gene Regulation

• MicroRNAs (miRNAs):– Short noncoding RNAs of ~22 nucleotides– Bind to specific regions of mRNA to alter

translation• Assist in cleaving the mRNAs • Or block the mRNA from translation

– ~1% of the human genome may encode miRNA!– Synthesized from larger precursors

• Processed by two endoribonucleases, Drosha and Dicer

Steps in miRNA Processing

• Long precursor pri-miRNA made in nucleus• Drosha and DGCR8 cleave pri-miRNA to a 70−80

nt precursor• Exportin and Ran export this precursor to the

cytoplasm• Dicer cleaves the pre-miRNA into dsRNA• Complement of miRNA removed by helicase• miRNA loaded onto protein complex such as RNA-

induced silencing complex (RISC)• RISC binds to target mRNA

RISC-miRNA prevents translation of mRNA

• (From previous slide): The miRNA sequence in RISC binds to complement in target mRNA

• If miRNA is ~ perfect complement, target mRNA is cleaved– Thus, the mRNA is not translated

• If miRNA is only partial complement, translation is blocked

How miRNAs are Processed to Prevent Translation

Ribozymes are RNA molecules that catalytically cleave RNA

• Cleave themselves or another RNA• 3-D structure integral to function• Inactive if denatured • Show Michaelis-Menton kinetics

– Saturable, have active site, have measureable KM, can be competitively inhibited

– Nucleophilic attack of sugar OH on phosphate (transesterification) followed by phosphodiester bond hydrolysis

Examples of Well-characterized Ribozymes

– Self-splicing group I introns• Example: 26S rRNA precursor from protozoan

Tetrahymena– RNase P

• cleaves precursors to tRNAs– Hammerhead ribozyme

• cleaves RNA of virusoids (circular RNAs that are replicated by plant viruses)

• So-named because 2 structure looks like head of hammer

Hammerhead Ribozyme

Cellular mRNAs are degraded at different rates

• RNA lifetime is one means of gene regulation• Half-lives vary from seconds to hours

– Typical vertebrate mRNA ~3 hrs– ~10 turnovers per cell generation– Shorter (~1.5 mins) half-lives for bacterial mRNAs

• Degradation via ribonucleases• Hairpin structures in mRNA can extend half-life

Retroviruses can make DNA from RNA

• Retroviruses have genomes of ssRNA and the enzyme reverse transcriptase– Virus enters host cell– Reverse transcriptase makes DNA from the

RNA• Then degrades the RNA from the DNA-RNA hybrid

and replaces it with DNA• DNA can then be incorporated into host DNA

Retroviral Infection of a Mammalian Cell and Integration into Host Chromosome

Retroviruses typically contain three genes plus a long terminal repeat

• gag (group associated antigen)– Encodes a long polypeptide that is cleaved into six smaller

proteins that make up viral core• pol

– Encodes protease that cleaves the long polypeptide, reverse transcriptase, and an integrase to insert DNA into host genome

• env– Encodes viral envelope

• Long terminal repeat (LTR) facilitates integration of virus genome into host DNA

Structure and Gene Products of an Integrated Retroviral Genome

Reverse transcriptases catalyze three reactions

1) RNA-dependent DNA synthesis2) RNA degradation3) DNA-dependent DNA synthesis

• Contain Zn2+, like DNA Pol• Use a primer of tRNA• Lack 3’ 5’-proofreading, like RNA Pol

- Make reverse transcriptase error-prone- Explains high rate of virus mutation/evolution

Some retroviruses cause cancer

• Some retroviruses contain an oncogene.– Example: Rous sarcoma virus has the src gene

• Src for sarcoma, a cancer of bone, fat, muscle, etc. (vs. cancer of epithelial cell origin)

• Encodes a non-receptor tyrosine kinase, an enzyme that affects cell division

Rous Sarcoma Virus Genome

HIV retrovirus causes AIDS

• HIV genome has genes for killing host (mostly T lymphocytes)– Results in suppression of immune system

• HIV-encoded reverse transcriptase is unusually error-prone– Complicates push for vaccine– At least one error per replication, so

potentially no two viral RNAs alike

HIV Genome

Pharmaceutical Targets for HIV (Antiretroviral Drugs)

• Reverse transcriptase inhibitors– Nucleotide or nucleoside analogs– Drug names tend to end in “dine” or “sine”

• Zidovudine (AZT), Didanosine (Videx), etc.

• Protease inhibitors– Since proteases used in cleaving proteins for

packaging into new viral particles– Drug names tend to end in “avir”– Indinavir, Saquinavir, etc.

Retrotransposons in eukaryotes have similarities to retroviruses

• Retrotransposons are mobile genetic elements in eukaryotes– Encode an enzyme with homology to reverse

transcriptase of retroviruses– Move between positions via RNA

intermediates• Using their enzyme to make DNA from RNA

– unlike bacterial transposons that move directly from DNA to DNA

More about Retrotransposons

• Lack the env gene so don’t form viral particles

• Examples: Ty element in yeast and Copia element in Drosophila

• See Fig. 26-36

Eukaryotic Transposons

Telomeres

• Are structures at the ends of eukaryotic chromosomes

• Have tandem repeats usually of T1-4G1-4

- With A-C on the opposing strand • Can be tens of thousands of bp long in

mammals• TG strand is longer than its complement,

leaves a 3’-overhang of several hundred bases

Telomerase extends the ends of linear chromosomes

• Telomeres are not easily replicated using DNA polymerases– Beyond an end there is no template for an RNA

primer– Chromosomes are shortened with each

generation• Telomerase adds telomeric sequences to

solve this problem

The Mechanism of Telomerase

• Telomerase has RNA with CyAx repeat to serve as template for synthesis of the TxGy strand of the telomere

• Telomerase binds to the 3’-end of the chromosome and hangs off so that the RNA template extends beyond it

• Telomerase extends the 3’-end, using the RNA of the enzyme as the primer

• The gap on the bottom strand is filled in by DNA polymerases

Telomerase Mechanism

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 requires processing before it becomes messenger 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: