Ribosome Assembly and Translational Regulation

What is the organization of genes in the E. coli chromosome?

Genes are encoded on both strands (top and bottom) of the DNA

Roughly equivalent number of genes on the two DNA strands

What is the most expressed genes by mass in bacteria?

90% of all RNA in E. coli is rRNA or tRNA

During rapid, expontential growth, how many ribosomes are made in each cell?

~70,000

How many rRNA operons are there in the E. coli’s chromosome?

7 nearly identical operons for rRNA production, rade of transcription is directional proportional to growth rate

Promoters of rrn operons have unique features that…

facilitate efficient transcription of the entire operon

What genes are encoded on the rrn operon in E. coli?

Contains genes encoding the three rRNAs and a tRNA (different one for each operon)

How many promoters are involved in the rrn operon?

Two promoters (P1 and P2)

Transcription initiation from both promoters is dependent on the housekeeping sigma factor 70

What transcriptional regulatory elements are there for P1?

upstream activation sequence (UAS), UP element, binding sites for FIS and H-NS

What are discriminators and how are they used in this operon?

3-nt sequence in the promoter

Consensus for non-rrn sigma 70 dependent promoters is three G’s on the non-transcribed strand (contributes to stability of RP_O)

In rrn: Pyrimidine (C and T)-rich instead of G-rich to destabilize the RP_O. Contributes to several properties of rrn promoters - transiotion to transcription elongation complex more readily, produce fewer abortive transcripts, inhibition by (p)ppGpp (stringent response)

What are nut-like sequences?

Participate in forming a transcriptional antitermination complex (TAC)

What are the rrn operons vulnerable to?

Termination by Rho due to their length and lack of translation

What do the TACs do?

Transcriptional antitermination complexes protect RNAP transcribing rRNA genes from pausing, as well as termination from hairpins and Rho

What does the rrn-TAC contain?

NusA, NusG, NusB, NusE (S10), ribosomal protein S4, and inositol monophosphatase (SuhB)

What is rrn-TAC structurally similar to?

lambaN-TAC

What do the TAC components do?

TAC components assemble on RNAP at the nut-like sites

TAC components effectively elongates the RNA exit channel, which aids in the proper folding of rRNAs

Eventually terminate transcription when they encounter the two tandem terminators (T1 and T2) at the end of the rrn operon

What processes rrn operon transcripts?

RNases take the one transcript, cut it into pieces, and release the mature mRNA pieces

What are the strategies that bacteria use to synthesize lots of ribosomes?

Location near OriC, 7 operons, TAC, multiple promoters

How does E. coli ensure that the 23S, 16S, and 5S rRNAs are made at the same levels?

TAC prevents early termination to ensure that the whole operon is synthesized

How do small non-coding RNAs regulate translation?

Small non-coding RNAs (sRNAs) have roles in regulating translation of certain mRNAs

sRNAs can either inhibit or stimulate translation of a targer mRNA

Binding of sRNA to target mRNA requires a RNA chaperone (ex. Hfg in E coli)

What is the stringent response?

conserved bacterial stress response that allows bacteria to adapt and survive under nutrient-limiting conditions

What does the stringent response involve?

Signal molecules guanosine pentaphosphate (pppGpp) and guanosine tetraphosphate (ppGpp) which are formed by the enzymes RelA and SpoT

How is ReiA activated?

In response to amino acid starvation

What occurs when uncharged tRNA-RelA complex bind to the A site?

Stimulates RelA activity leading to the transfer of pyrophosphate (gamma and beta phosphates) from ATP to GDP or GTP to form ppGpp and pppGpp, respectively

When is SpoT (p)ppGpp synthease activated?

In response to phopshate or fatty acid starvation

What does SpoT do?

Hydrolyzes (p)ppGpp to lower the level of (p)ppGpp when cells are not starved for phosphate or fatty acids

What occurs when (p)ppGpp binds to RNAP?

Depending on the promoter, binding of (p)ppGpp to RNAP either increases (ex. genes encoding amino acid biosynthetic enzymes) or decreases (ex. rRNA genes) transcription inititation

What is DskA

a small transcription factor working in conjunction with (p)ppGpp, binds the secondary channel (entry port for NTPs) of RNAP

Besides affecting transcription, what else does (p)ppGpp do?

inhibits DNA replication by inhibiting the activity of DnaG primase

When temperature changes, what happens to the RNA structures?

RNA structures can reversibly change dramatically with temperature changes (just like DNA)

What is a RNA thermometer?

When RNA strucutral changes occur due to temperature changes, it can regulate translation by changing access of the ribosome to the SD sequence when in 5’-UTR of the mRNA

At low temperatures, translation of what transcript is low?

rpoH which encodes sigma-32 heat shock factor

• Before 3’-end of rpoH transcript is made, the AUG start codon is accessible for translation initaition

• Once 3’ end of rpoH transcript is made, ribosome access to the AUG codon is blocked (very narrow window for tranlsation of rpoH transcript)

The actual SD sequence is NOT blocked by the transcript

Basal levels of sigma-32 are expressed due to the narrow window for translation of rpoH transcript

How does increased temperature increase the translation of rpoH?

• At 42 deg C, base pairing structure around start codon begins to break down in DB region

• Ribsome is able to place start codon in P site of 30S subunit to initiate translation

• Efficient translation leads to rapid increase in the level of RpoH protein in the cell, which can up-regulate transcription of heat shock operons