MT and CP

Question 1

mt genome

Answer 1

Circular, linear, fragmented
Trypanosomes maxi- and minicircles

Direct repeats - recombination, fragmentation - recombination.

Large variation in size, gene order due to intergenic regions

3-60 genes

Occurs in a complex with TFAM

Question 2

mitochondrial transcription

Answer 2

Phage-like polymerase, encoded in nucleus. Specificity factor provides specificity for promoters
Transcripts initiate at a conserved ‘a’ residue within recognition sequence

Question 3

mt post-transcriptional procecssing in yeast

Answer 3

tRNA in between mRNA or tRNA cistrons in unprocessed transcript

RNase P cleaves 5' of tRNA
RNase Z cleaves 3' of tRNA
CCA added onto 3' of tRNA
Cleavage at U-rich region on 3' of mRNA
Splicing

Question 4

Mt RNA processing in other organisms

Answer 4

Plants: Splicing, C–>U editing
ANimals: polyadenylation for stop codon
Trypanosomes: RNA editing by adding/removing UMP from mRNAs, from poly(U) tail of guide RNAs derived from minicircles

Question 5

Splicing in mitochondrial RNA

Answer 5

Yeast and plants, but not animals.

Type I intron: GTP and Mg, circular intron
Type II intron, Mg2+, lariat - adapted to spliceosomal machinery for nuclear genes

RNA maturases encoded within introns of Cyt b, that mediate splicing of Cyt B itself.

Question 6

Animal mt genome and transcription

Answer 6

Animal mitochondria have 3 promoters in the ‘D-loop’ - two transcripts from heavy-strand promoter, one from light-strand promoter

RNA Pol similar to yeast, with specificity factor

No splicing, but poly(A) to add stop codon.

Question 7

Translation in mitochondria

Answer 7

mt ribosomes and tRNA use vary across species

Sensitive to chloramphenicol but not cycloheximide

Yeast tRNA - wobble since only 25tRNAs encoded

Genetic code varies between species

Question 8

Transcription in chloroplasts

Answer 8

PEP - a2BB’B’’, with sigma factor encoded by nucleus - same mechanism of transcription as bacterial RNA Pol

NEP - Phage-like polymerase

Question 9

RNA Processing in chloroplasts

Answer 9

Cleavage at 5’-side to stability proteins by RNase J

Cleavage at 3’-side to secondary structure by PNPase

Cis-splicing to remove introns - type I and type II

trans-splicing to join ORFs that are separate on the genome (rbs12)

RNA editing C–>U

Polyadenylation promotes degradation

Question 10

Regulation of psbA translation

Answer 10

Light –> Reducing –> Breaks S-S bridge of cPABP –> cPABP binds to stem-loop on 5’-UTR –> promotes translation

Question 11

OM proteins

Answer 11

No N-terminal presequence
Single-pass a-helices can insert into OM directly via MIM complex

Porins are bound by hsp70, recruited to TOM70, pass through TOM40 into IMS, binds to SAM complex via B signal, inserts laterally into OM via SAM complex

Question 12

IMS proteins

Answer 12

No N-terminal presequence.

Translocate via TOM40, and folds in the IMS, preventing further translocation.

Mia complex facilitates S-S formation

Cyt c haem lyase inserts co-factor into Cyt C

(Cytb2 uses conservative sorting mechanism, cleaved by MPP in the matrix, IMP in the IMS)

Question 13

Metabolite carriers sorting

Answer 13

No n-terminal presequence

Hsp70, TOM70 –> Tom40 –> Tim9/10 chaperones in the IMS

TIM22 (voltage-dependent) –> IM carrier protein

Question 14

IM proteins with N-terminal preseq or matrix proteins

Answer 14

N-terminal presequence
Recognised by TOM20, translocates to TOM40, recognised by TIM50, translocates via TIM23.

Stop-transfer pathway for single-pass helical proteins

Conservative pathway for multi-pass helical proteins (and cyt b2, cleaved by MPP then IMP)

Matrix protein imported into matrix

All have N-term signal seq cleaved by MPP

Question 15

Important components of TIM23

Answer 15

PAM is the import motor - drives ATP hydrolysis via mtHsp70

TIM50 recognises presequence

Tim21 interacts with Tom22 - forming a substrate channel between the two.

Tim21 also interacts with Complex III and IV - ensures imported proteins are within vicinity of ETC

Question 16

Protein sorting in chloroplast

Answer 16

OM proteins - no presequence, inserts directly

IM proteins - presequence, TOC, TIC cleaved off in the stroma by MPP, stop-transfer or conservative

Stromal proteins - TOC, TIC, preseq cleaved off by SPP

Thylakoid membrane proteins - thylakoid-targeting internal sequence, remains in mature protein

Thylakoid lumenal proteins - bipartite sequence, one cleaved off in stroma by SPP, one cleaved off in thylakoid by TPP. Uses both Sec and Tat pathway for import into thylakoid.

Question 17

Yeast mitochondrial biogenesis

Answer 17

Haem is the O2 sensor

O2 –> haem biosynthesis –> HAP1 –> activates Cyt C transcription in the nucleus

HAP1 also activates expression of HAP2/3/4/5, which induces expression of small subunits of COX.

Question 18

Chloroplast biogenesis

Answer 18

Etioplasts convert into chloroplasts by:

Reduction in haem synthesis, which promotes chlorophyll synthesis from protoporphyrin IX.

LHC expression in the nucleus due to phytochrome activation, which degrades PIF repressors

Question 19

PRR proteins

Answer 19

RNA-binding proteins
Each repeat forms a double a-helix that binds a single RNA nucleotide, and facilitates correct processing of the RNA transcript