RNA Processing and Translation Flashcards Preview

HMS Chemical Biology and Biochemistry > RNA Processing and Translation > Flashcards

Flashcards in RNA Processing and Translation Deck (43)
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1
Q

Cental Dogma Steps

(in depth)

A
2
Q

RNA Processing outline

A
3
Q

largest known human gene

A

Human dystrophin

over 2.4 million bases

Requires ~16 hours to transcribe and is costranscriptionally spliced on the RBP1 platform

4
Q

RBP1’s role in transcription

A

Part of the RNA Pol II complex. Scaffold which contains a long C-terminal domain where splicing and processing enzymes dock.

Also associates with pRb

5
Q

pRb recruitment to a promoter. . .

A

blocks the assembly of pre-initiation complexes

6
Q
A
7
Q

RNA Processing CTD Steps

A

During the transition to elongation, the RNA polymerase becomes phosphorylated at specific sites. Initially, the polymerase is phosphorylated at position 5 of the repeat sequence; this recruits proteins involved in adding the 5’ cap to the mRNA (capping factors). Once capping has occurred, the polymerase becomes phosphorylated on both the 2 position and 5 position of the repeat, which helps recruit the splicing machinery. Near the end of transcription, the polyadenylation machinery (3’ end processing proteins) is recruited to the polymerase.

8
Q

5’ Capping

A

Functions of the cap:

Protects the 5’ end from degradation by nucleases Promotes mRNA export by binding export proteins

In cytoplasm, cap binding proteins are exchanged, and promote translation

9
Q

Chemistry of splicing

A
10
Q

Sequences Recognized in Polyadenylation

A
11
Q

Polyadenylation

A

Polyadenylation occurs through two steps: cleavage, followed by PolyA addition.

Poly A tails can be quite long, approximately 250 bases. Like the 5’ cap, the polyA tail also helps stabilize the mRNA (protects it from degradation by nucleases) and acts as a binding site for proteins that are involved in translation.

12
Q

Sequences Required for Splicing

A

Mutations at these sites can interrupt the process of splicing, generally leading to skipping of the exon next to which they occur. The branch point adenine is important for the mechanism of splicing.

13
Q

pre-mRNA

A

the name for the mRNA prior to completion of all of the processing steps

remains in the nucleus until it is processed

14
Q

Splice Mechanism

A

Ribonucleoprotein particles called snRNPs (pronounced “snurps”) recognize the sequences in the pre-mRNA and assemble together in a structure called the spliceosome, which catalyzes splicing. Each snRNP is composed of an snRNA (called U1, U2, U4, U5, U6) plus associated proteins. They are like the ribosome in that they are ribonucleoprotein particles that catalyze a reaction.

U1 snRNP recognizes the 5’ splice site, and U2 snRNPs recognizes the branchpoint and 3’ splice site.

Next, the U4/U5/U6 snRNPs join in, and then U1 and U4 are released.

The U2/U5/U6 complex caries out the splicing reactions, and then dissociates from the RNA.

Not shown here are so-called “exon-junction complexes” that load on and remain associated with the spliced mRNA; these proteins help facilitate nuclear export of the spliced mRNA.

15
Q

snRNP structure

A

Each snRNA contains a similar sequence called an Sm motif that recruits an Sm protein-this protein is present in all snRNPs (U1, U2, U4, U5, U6). Each snRNP carries a small nuclear RNA (snRNA, U1 snRNA, U2 snRNA, etc) as well as proteins that are unique to each snRNP. The protein components of the U1 and U2 snRNPs are shown above. The snRNAs base pair with the conserved sequences in the 5’ splice site and 3’ splice site; U2 snRNA recognizes the branch point sequence to help bulge out the adenosine that will perform the first step of splicing.

16
Q

Intron vs Exon definition

A

Intron definition was an old theory, but could not be true as exons on the end would be lost. Exon deifnition is now the accepted true model.

Exons contain specific sequences called “exonic splicing enhancers”, or ESEs, that recruit SR proteins (they are rich in Serine (S) and Arginine (R)). The SR proteins help recruit the U1 and U2 snRNPs to the proper sites at each end of the exon. Once two neighboring exons are defined, the U1 snRNP interacts with the U2 snRNP from a different exon to initiate splicing by recruitment of U4/U5/U6. The orange protein is called U2AF for U2-associated factor; it acts as a bridge between the SR protein and the U2 snRNP.

17
Q

How can weakly-binding splice factors be effective in efficient, selective, high-affinity splicing?

A

Works because protein complexes that help define the exon are cooperative. They each bind only weakly, but their net affinity is summative. The flip side of this principle is that if a single binding sites is mutated, it can lead to failure to define the exon, and as a result the exon will be skipped in the splicing process.

18
Q

Why is exon definition important?

A

In higher eukaryotes, exons are small and introns are very large.

Because the sequences that promote splicing are short, the introns often have sequences that look like splice sites, but it is important that these “decoy” splice sites not be used. By including the exon in the recruitment process, the proper splice sites can be selected.

19
Q

What type of mutation would cause each of these splice misfunctions?

A

In case B, the ESE in exon 2 has been mutated.

In rare cases, sometimes loss of a sequence will lead to use of a “weaker” sequence nearby; this can lead to extension of an exon (C) or even the creation of new exons (D). In this case, imagine a mutation that creates a new binding site for an SR proteins that is located between two decoy splice sites. This pathway of de novo exon inclusion is thought to be one pathway by which new protein sequences can evolve.

20
Q

Example of extensive alternative splicing

A

alpha-tropomyosin

There are two basic explanations for protein isoforms: 1) gene duplication and divergence; 2) alternative splicing. Thus some proteins that have different isoforms are encoded by different genes. However, most isoforms arise by alternative splicing

21
Q

Types of alternative splicing

A
22
Q

RNA Export

A

Many of the proteins loaded onto the pre-mRNA as it becomes a mature mRNA are important for efficient export from the nucleus. These include the EJC (exon junction complex) proteins that are loaded on during splicing, as well as SR proteins that bind to exons. Proteins that bind to the cap are also important for nuclear export; these are exchanged for translation initiation factors in the cytoplasm.

23
Q

“Pioneer” Round Translation

A

The first time the ribosome encounters the mRNA is called the “pioneer” round of translation, and is a bit different from subsequent rounds of translation (bulk translation). During this first round of translation, the ribosome has to “bump off” any other proteins bound to the mRNA, including the exon-junction complex proteins that remain on the mRNA (labeled EJC core) above. These complex are left as marks on the mRNA at each exon-exon junction (they essentially mark where the splice sites occur).

24
Q

Overall translation error rate

A

~1/10,000 amino acids

25
Q

Translation overview

A
26
Q

Ribosome structure

A
27
Q

Ribosomal subunit rRNAs

A
28
Q

Steps regulated to ensure translation fidelity

A

The amino acyl-tRNA synthetase needs to select the proper amino acid.

The synthetase needs to select the proper tRNA.

The proper charged tRNA must recognize the proper codon during translation. Note that it is the ribosome that is responsible for ensuring that the right amino-acyl tRNA is paired up with the right codon during translation.

29
Q

tRNA structure

A
  • Each is a single chain of between 73-93 ribonucleotides that folds into an L-shaped 3- dimensional structure by intramolecular base pairing
  • They are modified following transcription to include several unusual bases such as Ψ (pseudouridine). These modifications enhance the structural diversity of tRNAs, facilitating specific recognition by a particular amino-acyl tRNA synthetase
  • About half of the nucleotides are base-paired to each other, with the exception of those in the anticodon and the amino acid acceptor arm
  • The 5’ end is phosphorylated
  • The amino acid is attached to the 3’ hydroxy group of the ribose at end of the tRNA, and the tRNA always ends in the sequence ACC
  • The anticodon is the region of the tRNA that will base pair with the codon in the mRNA. The anticodon loop is positioned far away from the acceptor stem, which will carry the activated amino acid
30
Q

tRNA Synthetase Proofreading

A

The editing site will bind and hydrolyze (release) an amino acid that is not the proper amino acid.

In the case of the isoleucine-tRNA synthetase, the editing site will bind valine that is incorrectly attached to the tRNA. Note that isoleucine is larger, and it cannot fit into the editing site. Thus, if the proper amino acid is attached, isoleucine, it will not be edited by the tRNA synthetase.

31
Q

Translation Initiation

A

Note that the names of eukaryotic initiation factors begin with “e” for eukaryotic.

A preinitiation complex is formed between initiation factors, the 40S ribosome, and the met-tRNA initiating amino-acyl tRNA. eIF2 helps deliver this aminoacyl-tRNA to the preinitiation complex. eIF1A helps the preinitiation complex assemble.

This preinitiation complex then binds to the 5’ end of the mRNA. This is mediated by a protein complex called eIF4; one protein in the complex called eIF4E binds to the 5’ cap of the mRNA.

Once the initiation complex is loaded, the 40S subunit of the ribosome then scans down the mRNA until it finds the first AUG. One protein in the eIF4 complex helps unwind the mRNA. But some mRNAs contain proteins that bind to hairpin structures in the 5’ untranslated region of the mRNA-this can serve to negatively regulate (inhibit) translation -> Attenuation

Once the 40S subunit finds the first AUG, other initiation factors dissociate, and the 60S (large) subunit binds, forming the full ribosome, which can now carry out the elongation step of translation

32
Q

Translation Elongation

A

Translocation requires the protein EF2 (similar to EF-G in bacteria). Again, translocation requires hydrolysis of GTP.

33
Q

Peptide bond formation reaction

A

Peptide bond formation is catalyzed by an adenine base in the 28S ribosomal RNA from the large subunit, in a mechanism similar to that of serine proteases. Thus the large subunit contains the peptidyl transferase activity. This activity is a target of some antibiotics.

34
Q

Translation Termination

A

Termination also involves the use of specific termination factors that bind to stop codons. This step also involves GTP hydrolysis.

35
Q

Polysome formation

A

One important mechanism for stimulating translation in eukaryotes is polysome formation. In this case, proteins that bind the polyA tail (called poly-A binding proteins or PABPs) also interact with the initiation factors that bind to the 5’ cap. This forms a stable complex, which allows reassociation of 40S subunits with the initiation factors after they have dissociated from the mRNA following a round of translation of the mRNA. These structures can be visualized in the electron microscope

36
Q

NMD occurs. . .

A

during translation

37
Q

When are EJCs removed?

A

During translation. This means that they remain attached as the transcript leaves via the nuclear pores.

38
Q

Order of events in mRNA processing

A

5’ cap → splicing → poly A tail

39
Q

In translation, what is the role of the large vs small subunit?

A

Small subunit threads mRNA through, the large subunit does the stuff

40
Q
A
41
Q

SRP

A

Signal Recognition Protein

Recognizes the ER signal seq and brings it to an ER membrane translocon.

42
Q

Signal Peptidase

A

Signal peptidase cleaves ER signal sequences as the protein is being translated via the translocon into the ER.

43
Q

ERO1

A

Re-oxidizes PDI