Molecular Bio - Final (New content)

New content post-exam 2 Nov 3rd onward

Introns

non-coding sequences in pre-mRNA

Exons

sequences in pre-mRNA that code for proteins

What is step one of intron removal?

Break sugar phosphate bonds at intron boundaries

What is step two of intron removal?

Rejoin free ends from intron removal into a continuous mRNA molecule

What are the most frequent class of introns?

GU-AG intron group (class I), associated with eukaryotic nuclear genes

Where are intron groups I, II, III found?

Found in mitochondria and chloroplasts (contain machinery necessary for transcription/translation)

pre-tRNA intron

Found in tRNA nucleus

Splicesome

Splicing machinery consisting of snRNA (5 per cell)

What type of RNA is processed by U3 snoRNA in the nucleolus?

rRNA

What are snRNP (snurps, U1-U6) made of?

snRNA and protein

Function of snRNP?

recognizes 5’, 3’, and branch sites on the pre-mRNA

pair with specific intron segments and assemble spliceosome

What does U1 snRNP recognize?

5’ splice site

What does U2 snRNP bind to?

branch site

What does protein U2AF bind to?

3’ splice site

What does U4 and U6 snRNP bind to?

U2 snRNP

What does U5 bind to?

First downstream exon

What happens in step one of splicing?

• intron and exon are cut at 5’ splice site after binding of U1

• using U5, the free 5’ end loops around and joins adenine to make lariat intermediate

What happens in step two of splicing?

free 3’ end of upstream exon displaces the intron lariat structure, two exons are joined together (transesterification)

What do eukaryotic genes with long introns have?

Exonic enhancer sequences with the exon that help to position splicing machinery

Self splicing

Removal of an intron using ribozyme activity of the RNA molecule, does not require snRNP’s

Ribozyme

RNA molecule that has enzyme activity

Which intron groups are self splicing?

I, II, III — due to endosymbiont theory

What technique has been used for determining intron and exon boundaries?

Electron microscopy, R-loop analysis

Alternative splicing

Alternative ways to make two or more final mRNA molecules using exon sequences from same gene

Advantages to alternative splicing?

Saves space, increase protein diversity

Alternative promoter selection

two alternative promoters are available, choice depends on transcription factors specific to type of cell

Alternative tail site selection

alternative sites for adding poly(A) tail are possible, choice depends on cell type

Alternative exon cassette selection

Choice between splice sites, depending on choice a particular exon may or may not appear in final mRNA

What affects choice in alternative exon cassette selection?

• Proteins recognizing splicing enhancers and silencers at splice sites

• Developmental stages and tissue types influence regulation

Proteins that recognize enhancers?

SR proteins

Proteins that recognize silencers?

hnRNP

Where is cell signaling important?

In activating proteins that bind to splicing enhancer/silencer

Intron retention

Intron is retained in coding sequence

Pathways of intron retention

1. degraded in nucleus

2. degraded in cytoplasm

How does intron degradation in cytoplasm occur?

1. exonucleases

2. miRNA: non-coding micro RNA, binds to retained introns and degrades dsRNA

3. NMD: surveillance system that destroys defective mRNA with premature stop codons or long 3’UTR

What causes defective mRNA molecules?

Nonsense mutation

Overall, what are the two functions of miRNAs?

1. Mark defective RNA for degradation

2. Silence mRNA post-transcription

What must mRNA have in order to exit the nucleus?

Cap, tail, and introns removed

Exportins in nucleus do what?

bind to the mRNA and pore complex

What is needed for mRNA to be transported through the nuclear pore?

ATP hydrolysis

Nuclear pore complex

Cluster of 30 nucleoporins that control traffic to the nucleus, largest protein complex found in cell

Why might a cell need to degrade mRNA?

• Structural defects in mRNA

• Limit excess protein production

• mRNA no longer needed

How does degradation of mRNA occur in eukaryotic cells?

Poly(A) tail and 5’ cap are removed, then exonuclease degrades mRNA in the 5’ to 3’ direction

What controls mRNA stability in a cell?

• stem loops in 5’ and 3’ UTR block exonuclease from degrading ends

• destabilizing sequences in UTR (ex - AU rich)

Trans splicing

Splices together segments from two different primary transcripts (rare, found in Trypanosomes)

Benefit of trans splicing?

Allow pathogenic organisms to evade detection by immune system

Protein splicing

intervening sequences can be spliced from protein sequences after translation, inteins and exteins are equivalents of introns and exons (yeast, algae, bacteria)

Advantage to protein splicing?

Allow for specialization, aid in conformation of protein, evade immune detection

Translation

Synthesis of proteins in cytoplasm and ER, may also occur in mitochondria and chloroplasts

Steps of translation?

initiation, elongation, termination

Where are ribosomes made in the cell?

nucleolus

Peptide bonds

link amino acids

During translation, the bases of mRNA are read…

in groups of 3, codons

Each codon represents…

an amino acid

Degeneracy of the genetic code

Some amino acids are encoded by more than one codon

How many stop codons are there?

3 (UAA, UAG, UGA)

Start codon

AUG, encodes for Met

How does tRNA read the codons?

It recognizes codon on mRNA using the complimentary anticodon in its own structure

tRNAs can read more than one codon, BUT…

the codons must all code for the same amino acid

Which codons are complimentary in the binding of tRNA to mRNA?

The first two

Wobble hypothesis

base at 5’ end of tRNA anticodon is not as spatially confined as the other two bases, allowing tRNA to recognize multiple codons

aminoacyl tRNA synthetase enzymes are responsible for…

activating or charging tRNA via amino acid attachment

Reading frames

bases of mRNA are read in groups of three, with each codon corresponding to one amino acid

How does ribosome know where to bind to the transcript?

Recognizes the AUG

Open reading frame

any sequence of DNA or RNA beginning with a start codon

initiator tRNA

charged with methionine and binds to the start codon to initiate protein synthesis

Why would initial methionine be removed later on after synthesis of a protein?

signal sequence that direct proteins to specific locations is removed once a protein gets to a specific location (IDFK GOOGLE THIS SHIT BRUH)

Ribosomes are exported from…

the nucleus through the nuclear pores on the membrane

Why would a single mRNA have multiple reading frames?

to code for multiple proteins

Ribosomes are composed of…

a large and small subunit that join together to move across mRNA

Eukaryotic ribosomes are ___ S, with ___ S and ___ S subunits

80, 60, 40

Differential velocity centrifugation

used to purify organelles based on size

Ribosomes are complexes of…

rRNA and ribonucleoprotein

Ribosomal proteins are produced in the ___ and transported through the ___ into the ___

cytoplasm, nuclear pore, nucleolus

rRNA is produced in the ___ and assembled with ___ to form the ___

nucleolus, ribosomal proteins, large and small ribosomal subunits

Exportin proteins do what?

interact with ribosomal subunits and nucleoporins to help with nuclear export

What provides energy for the ribosome to move along the mRNA?

GTP hydrolysis

Function of small ribosomal subunit?

Scans the mRNA, reads codons

Function of large ribosomal subunit?

Links the incoming amino acid to the growing protein chain

Free ribosomes

free to move anywhere in the cytoplasm, synthesize structural proteins

Membrane bound ribosomes

bound to endoplasmic reticulum, synthesize proteins to be secreted

What types of proteins are synthesized in mitochondria and chloroplasts?

proteins needed for cellular respiration or photosynthesis

Initiation factors

13 proteins needed to initiate translation

Cap binding complex

attaches to the 5’ cap, consists of RNA helicase and scaffold protein

Pre-initiation complex is composed of…

43S complex + ternary complex

Cap binding protein causes mRNA to…

change its conformation into a ringed structure, allowing for the 43S complex to bind

PABP protein binds to…

the polyA tail

If mRNA is single stranded, why is helicase activity necessary for initiation of protein synthesis?

5’ UTR contains stem loop structures to protect mRNA from degradation

Why is it important that eIF4A has helicase activity?

so it can use ATP and hydrolysis to unwind stem loop structures in the 5’ UTR

What does eIF4G protein do?

scaffold protein that interacts with polyA binding protein PABP

What two things are needed to recruit mRNA to initiation machinery?

eIF4A and eIF4G

Role of eIF3 in pre-initiation complex?

helps to position small ribosome subunit at start codon AUG

Role of eIF2 in pre-initiation complex?

• brings charged tRNA to ribosome

• helps in scanning mRNA for start codon

• regulate protein synthesis in response to environment

• uses energy from GTP

How does phosphorylation affect protein synthesis?

it slows it down

Once AUG has been found, which subunit will bind?

60S

If there are multiple AUG’s, how will the small subunit decide where to bind?

it will bind to the first AUG with a regulatory sequence around it

A site

binds aminoacyl tRNA (tRNA bound to an amino acid)

P site

binds peptidyl RNA (tRNA bound to the protein being synthesized)

E site

binds to free tRNA before it exits the ribosome

What moves the tRNAs from site to site?

elongation factors and hydrolysis of GTP