06 - Splicing & Exporting RNA & Reading the Genetic Code

• To create a protein, a eukaryotic gene must be:

Transcribed into RNA (primary transcript)

• Pre-mRNA is processed (including splicing)

• Exported from the nucleus

• Translated by ribosomes

RNA splicing

removes introns from pre-mRNA

relative size comaprison between exon and intron

• Introns are usually much larger than exons

“lariat”

• Each splicing event removes one intron as a “lariat”

This is a complex process carried out by 5 snRNA (small nuclear RNA) molecules and 100’s of proteins!

Three types of splicing sequences in the pre-mRNA base-pair with snRNA

5’ splice site (begin), 3’ splice site (end), and a branch point

Splicing of eukaryotic mRNAs

1. U1 snRNP binds the 5’ splice site. Proteins bind the other sites

2. U2 snRNP binds the branch point site, which causes an adenosine to bulge out

3. A U4/6 pair and U5 snRNPs enter and U6 replaces U1 at the 5’ splice site. U4 and U1 are released.

4. The branch point adenosine attacks the 5’ splice site and forms a new covalent bond (and loop)

5. The newly created 3’OH from the 5’ splice site then reacts with the 3’ splice site to join the exon sequences and release the lariat

6. Exon junction complex (EJC) marks newly completed splicing event

spliceosome complex

The 5 snRNAs (called U1, U2, U4, U5, and U6) complex with proteins to form snRNPs (“snurps”). snRNPs together form the spliceosome complex

catalytic site

The snRNAs form the catalytic site of the splicesome and create it new (de novo) on each mRNA molecule

What property of the snRNAs is critical in selecting splice sites and carrying out the splicing process!?

their ability to base-pair with specific sequences in the pre-rna

Splicing Errors

Some of the most common are skipping an exon and mistakenly selecting a “cryptic splice site” due to similarity to a splice site consensus sequence.

what can help decrease spiling errors

Proteins help mark splice sites for snRNP recruitment to decrease the frequency of these errors (and help regulate alternative splicing)

Could a point mutation/SNP impact splicing? How?

yes because it would mutate consensus sequences and then mRna wouldn’t reconize them as much

Consequences of errors in splicing

Mutations at splice sites can prevent excision or alter splicing, potentially resulting in altered amino acid sequence after translation and:

• mRNA degradation

• Accumulation of unspliced intermediates

• Altered mRNA regulation or transport

what contributes to a large amount of total RNA

mRNA and the pre-mRNA precursor constitute a small percentage of total RNA (up to 10%) of most eukaryotic cells, but a large percentage of RNA being synthesized by the cell at any given time

is everything that gets transcribed, get tranlsated

A lot of material is transcribed that the cell would NOT want to translate! (ex. pre-mRNAs, excised introns, broken/damaged RNA, accidentally transcribed non-coding regions, noncoding RNA)

Degradation of RNA

The default fate of RNA in the nucleus is degradation in the RNA exosome by RNA exonucleases.

But: Proteins binding to the RNA can protect it (& signal that it is ready for function/export)

Selective nuclear export of mRNA

Nuclear transport receptors help guide completed RNA to rapidly pass through channels in the nuclear membrane created by nuclear pore complex proteins

Completion of processing can be detected by what proteins are bound to the mRNA

What types of proteins/complexes would be associated with an unfinished pre-mRNA?

the enzymes eg capping enzyme, rna polymerase

Translation of mRNA is catalyzed by

ribosomes

ribosomes

Ribosomes are protein synthesizing machines that direct the elongation of polypeptides

They are the most abundant RNAprotein complexes in the cells and require energy from GTP hydrolysis.

Ribosomes in the cytoplasm are “free” or on ER

is the Structure of ribosomes conserved

highly conserved = highly important!

S value (Svedberg unit)

measure of the sedimentation rate during centrifugation – affected by both mass and shape

the relation of size of molecule, s value and sedimentation

The larger the molecule, the larger the S value, the faster the sedimentation

parts of ribosome

60S large subunit of ribosomes

40S small subunit of ribosomes

what makes up the 60S large subunit of ribosomesand 40S small subunit of ribosomes

• 28S, 5.8S, 5S make up 60S large subunit of ribosomes

• 18S makes up the 40S small subunit of ribosomes

• More than 80% of the RNA in most cells is ____

rRNA!!

There are multiple copies of rRNA genes in the genome (a type of moderately repetitive DNA). Why is that necessary?

aplification of rna → protein

Synthesis of ribosomal RNAs (rRNAs)

rRNA is made from rDNA (rRNA genes), which is found in clusters of repeats in specific regions of the genome

during interphase, rDNA clusters come together in irregularly shaped nuclear structures

_____ are sites of ribosome biosynthesis

Nucleoli

inside the Nucleoli

kinda like a christmas tree

• Black dots on base of the “branches” (middle of tree) are RNA polymerases on DNA template

• The “branches” are the rRNA transcripts

• Dots at the end of the branches are thought to be the start of ribosome assembly

The 5.8S, 18S, and 28S rRNAs are made from

a larger precursor rRNA transcribed by RNA polymerase I in the nucleolus

5S rRNA is made by

RNA polymerase III (not in the nucleolus)

Processing of precursor rRNA

begins during transcription:

• chemically modified at specific positions

• cleaved into smaller rRNA (28S, 18S, 5.8S)

5S is made separately and does not need chemical modification

Chemical Modification of rRNAs

• Isomerization → some uridines are modified to pseudouridine

• Methylation → methyl group attached to the 2’OH of the ribose

• May aid in ribosome assembly/function

Processing requires

small, nucleolar RNAs (snoRNAs) associated with proteins to form snoRNPs (small, nucleolar ribonucleoproteins)

snoRNA come from

introns transcribed by RNA pol II

Producing ribosomes

Production of rRNA and assembly with proteins into the ribosome subunits occurs in the the nucleolus

Large and small subunit join together on the mRNA molecule in the cytoplasm

Which components are produced in the nucleolus?

Which need to be brought in?

5.8s 18s 28s rnas

everything else needs to be brought in

Function of tRNAs

tRNA are responsible for “decoding” the mRNA to create proteins

Different tRNAs are modified to carry different amino acids.

Structure of tRNAs

• Four double-stranded stems and three single stranded loops = cloverleaf structure

• Contains unusual bases that have been posttranscriptionally chemically modified by enzymes.

• Loop regions create binding sites for specific proteins

• 3’ end is the amino acid acceptor arm

• tRNA “clover leaf” secondary structure folds into a L-shape tertiary structure

• Anticodon loop is at one end of the Lshaped molecule and the 3’amino acid acceptor arm is on the opposite end.

Will all tRNA (carrying the different amino acids) have the exact same tRNA nucleotide sequence?

no because they have to be different

Transcription & processing of tRNA (transfer RNA)

• tRNAs are encoded by tDNA sequences located in small clusters of repeats scattered around the genome

• Different types (species) of tRNA, each ~80 nucleotides in length

• Transcribed by RNA pol III

• Processing: tRNA precursor is trimmed and bases are modified

what is required to make a polypeptide

rRNA, tRNA , and mRNA

- Catalysis, decoding, and information storage

- All use the super power of base-pairing

codons

three nucleotides

• Codons are written 5’ to 3’:

what makes up a amino acid

• A set of three nucleotides (a codon) ‘codes’ for a single amino acid

How is the genetic code “read”?

The genetic code is read in codons that are non-overlapping and consecutive (sequential with no skips/gaps)

frameshift mutations

Mutations that change which reading frame is used are called frameshift mutations

codon/anticodon base-pairing

tRNAs decode the mRNA message through codon/anticodon base-pairing

stop or termination codons

signal the ribosome to stop reading the mRNA

The genetic code is said to be degenerate (redundant) because

more than one codon can encode the same amino acid

silent mutation

alters the nucleotide sequence, but still encodes the same amino acid (vs missense mutation)

nonsense mutation

A mutation that leads to a stop/termination codon is called a

What would happen if a single nucleotide was removed or added in the coding sequence?

point muttaion

What do you notice about codons that encode the same amino acid?

they have the first 2 bases but the last one differs

What do you notice about codons that encode amino acids with similar properties?

first two positions common

Why might this be beneficial (why would the genetic code have evolved this way)?

point mutaions are mostly silent

tRNAs: Wobble Base-Pairing

tRNA are designed so that the codon/anti-codon pairing in the first two positions is strict but the third is flexible (can “wobble”)

Wobble base-pairing explains why many codons for the same amino acid differ only in their third (“wobble”) base

How is the correct amino acid added to each tRNA?

“Charging” a tRNA: Appropriate amino acid must be covalently linked to the free OH on the adenosine at the 3’ end of the tRNA (amino acid acceptor arm)

what catalyzes a charging tRna

Aminoacyl-tRNA synthetase is the enzyme that catalyzes charging a tRNA with an amino acid

Typically, 20 different aminoacyltRNA synthetases; 1 per amino acid.

(some bacteria have found ways to get around this and have slightly fewer

“Charging” tRNA with an amino acid process

Aminoacyl-tRNA synthetase interacts with tRNA to bind and recognize both the anticodon loop and the amino acid acceptor arm

2 step reaction adds the amino acid to AMP (“activation”) and then transfers it to the tRNA

Proofreading the charging of tRNAs

• Editing site of the aminoacyltRNA synthetase is used for proofreading: Only incorrect amino acids will fit in the editing site and will be cleaved by hydrolysis

Error rate in tRNA charging is 1 per 40 000

Way higher than for DNA replication…How is that ok??

the proteins get turned over and degraded

Ensuring accuracy of tRNA charging and translation:

• Correct amino acid has the highest affinity for the synthetase active site

• Aminoacyl-tRNA synthetase has proofreading/editing function

• During translation, the codon/anti-codon pairing selects the correct tRNA

which end is amino acids added

• Amino acids are added to the C-terminal end of a growing polypeptide chain

where does decoding of mrna take place

• This decoding of the mRNA message into protein occurs in ribosomes

Structure of ribosome

• The interface between small and large subunits forms a spacious cavity

• mRNA molecule is ‘threaded’ through the small subunit

• Polypeptide is exits via a tunnel through the large subunit

ribozymes

RNA that has catalytic activity

example of ribozymes

snrna

rrna

Small ribosomal subunit

is important for decoding mRNA (ensuring correct codon/anticodon pair)

Large ribosomal subunit

is important for catalyzing peptide bond formation to add a new amino acid to the growing polypeptide chain

Which of the following are true about non-coding RNA in eukaryotes?

A) They are all synthesized by RNA polymerase II

B) rRNA is non-coding RNA that functions in ribosomes

C) snRNA is a non-coding RNA involved in RNA splicing

D) TFIIH is a non-coding RNA involved in transcription initiation

E) Some non-coding RNA are encoded by many copies in the genome

F) All rRNA are transcribed in the nucleolus

G) tRNA are transcribed in the cytoplasm

H) snoRNA are involved in rRNA processing

b,c,e,h

Which of the following are required in the nucleolus? Review:

A) snoRNA

B) Aminoacyl tRNA synthetase

C) Splicesome

D) mRNA

E) RNA pol I

F) RNA pol III

G) 5S rRNA

H) 18S rRNA

I) Ribosomal proteins

a,e,g,h,i

Which of the following are true about tRNA?

A) Their function is performed in the nucleus

B) Cells must have a different tRNA species to match every possible codon

C) The amino acid acceptor arm will interact with mRNA

D) They are made from larger RNA precursors

E) They are translated into proteins

F) 1 tRNA species could decode multiple codons because of wobble base-pairing

G) They are chemically modified to contain unusual bases

d,f,g

Which of the following are true about aminoacyl-tRNA synthetase?

A) They transcribe tRNA

B) They catalyze adding an amino acid to tRNA

C) They produce ATP

D) They can differentiate between different tRNA

E) They can tell if the correct amino acid has been added to a tRNA

F) They translate mRNA into protein

b,d,e

Which of the following are typically true about aminoacyl-tRNA synthetase?

A) There is a different aminoacyl-tRNA synthetase for each different amino acid

B) There is a different aminoacyl-tRNA synthetase for each different tRNA

C) There is a different aminoacyl-tRNA synthetase for each different codon

D) There is only 1 type of aminoacyl-tRNA synthetase for charging all the different tRNA

E) There are more different codons than there are different aminoacyl-tRNA synthetases

F) Aminoacyl-tRNA synthetase adds an amino acid to the anti-codon of a tRNA

a,e