ch 8 micro

Each segment of double-stranded DNA contains how many possible open reading frames? one three four six

6

Template strand

DNA stand that specifies the base sequence of the new complementary RNA

unsticking stuck ribosomes

The molecule tmRNA( and protein tagging) has properties of both tRNA and mRNA. It rescues ribosomes stuck on damaged mRNA that lacks a stop codon.

Export of Prefolded Proteins to Periplasm

The twin arginine translocase (TAT) can move a subset of already folded proteins across the inner membrane and into the periplasm. ---Powered by the proton motive force

sigma factor

factor helps the core enzyme detect the promoter, which signals the beginning of the gene. Recognizes consensus sequences at the -10 and -35 positions, relative to the start of the RNA transcript (+1). --- Ecoli sigma -70. single bacterial species can make several different sigma factors. ( based on specificity like thermo)

RNA stability is measures in term of what? average?

half-life. -The average half-life for mRNA is 1-3 minutes -The stabilities of the different kinds of RNAs differ drastically

Initiation of protein synthesis

initiation factors help binding of mRNA to ribosome. the first AA that come in the Psite. once the initations factors leave their a GTP use

RNA polymerase

is a complex enzyme that carries out transcription by making RNA copies (called transcripts) of a DNA template strand.

in bacteria RNA poly I

is a holoenzyme and composed of -Core polymerase: a2, b, b′ Required for the elongation phase -Sigma factor: sigma sign Required for the initiation phase; joints the poly - holoenzyme does not function by itself

Elongation of protein synthesis

once another tRNA binds like in the initiation step then the peptidyltransferase activity makes a peptide bond between the AA on peptide in the P site and the AA in the Asite. the formation results in the AA chain transfer to the A site the shift is done by the GTP hydrolyzis.

coding and template strand

the coding strand is the same has mRNA and the template strand is backwards.

Elongation of RNA transcripts

-Elongation is the sequential addition of ribonucleotides from nucleoside triphosphates. -The original RNA polymerase continues to move along the template, synthesizing RNA at ~ 45 bases/sec. -The unwinding of DNA ahead of the moving complex forms a 17-bp transcription bubble. -Positive supercoils ahead are removed by DNA topoisomerases.

protein folding

-Folding of many proteins requires assistance from other proteins called chaperones. Folding can be controlled by the amino acid sequence. GroEL and GroES chaperones --Form stacked ring with a hollow center --The protein fits inside the open hole DnaK chaperones --Do not form rings --Clamp down on a polypeptide to assist folding

Peptidyl transferase catalyzes the movement of the peptide from the tRNA in the _______ to the tRNA in the _______. a) P-site; A-site b) A-site; P-site c) P-site; E-site d) E-site; A-site e) E-site; P-site

A

The sigma factor is directly required for a) Transcription initiation b) Transcription elongation c) Translation initiation d) Translation elongation

A

Similar genes within the same organism that have different functions are called a) Homologs b) Orthologs c) Paralogs d) Homogeneous e) Synonymous

C

_______ are protein-degrading organelles found mainly in eukaryotes and archaea. a) Lysosomes b) Proteases c) Proteasomes d) Degradosomes e) Degrons

C

The Genetic Code

Consists of nucleotide triplets called codons --There are 64 possible codons: -61 specify amino acids ---Include the start codons -3 are stop codons The code is degenerate or redundant ---Multiple codons can encode the same amino acid The code operates universally across species ----Remarkably, with very few exceptions --wobble one anticodon to pair with different codons that differ in the third nucleotide

The termination of transcription can be caused by which of the following? a) Stem-loop structure b) Stop codon c) Rho factor d) Two of the above e) All of the above

D

Homologs, Orthologs, and Paralogs

Genes that are homologous likely evolved from a common ancestral gene. Orthologous genes --Genes duplicated via appearance of a new species ---Have identical function in different organisms Paralogous genes ---Genes duplicated within a species ---Have slightly different tasks in a cell

bacteria and outer membrane

Gram-negative bacteria need to export proteins completely out of the cell. ----For example, digestive enzymes and toxins Seven elegant secretion systems have evolved: -Labeled Type I-VII ---Some deliver the exported proteins to other dedicated transport proteins in the periplasm. ---Others provide nonstop service.

Bacterial transcription and translation are coupled

-Different ribosomes can bind simultaneously to the start of each cistron within a polycistronic mRNA. -Before RNA polymerase has even finished making an mRNA molecule, ribosomes will bind to the 5′ end of the mRNA and begin translating protein. ---This is called coupled transcription and translation. -Eukaryotic microbes, on the other hand, use separate cellular compartments to carry out most of their transcription and translation.

Attaching Amino Acids to tRNA

-Each tRNA must be charged with the proper amino acid before it encounters the ribosome. -The charging of tRNAs is carried out by a set of enzymes called aminoacyl-tRNA synthetases. ---Each cell has generally 20 of these "match and attach" proteins, one for each amino acid. -Each aminoacyl-tRNA synthetase must recognize its own tRNA but not bind to any other tRNA. ---So each tRNA has its own set of interaction sites that match only the proper synthetase. THIS IS ATP dependent processs

different classes of RNA. mRNA, rRNA, tRNA, sRNA, tmRNA, catalytic RNA

-Messenger RNA (mRNA): encodes proteins -Ribosomal RNA (rRNA): forms ribosomes -Transfer RNA (tRNA): shuttles amino acids -Small RNA (sRNA): regulates transcription or translation ---the smallest, important in regulation on the organism in stress -tmRNA: frees ribosomes stuck on damaged mRNA --Dual tRNA-like and messenger RNA -like properties -Catalytic RNA: carries out enzymatic reactions

Translation of RNA to Protein

-Once a gene has been copied into mRNA, the next stage is translation, the decoding of the RNA message to synthesize protein. -An mRNA molecule can be thought of as a sentence in which triplets of nucleotides, called -codons-, represent individual words, or amino acids. Ribosomes are the machines that read the language of mRNA and convert, or translate, it into protein. They do so via the --genetic code.

Protein secretion: cell membrane

-Proteins destined for the bacterial cell membrane or envelope regions require special export systems. -Proteins meant for the cell membrane are tagged with hydrophobic N-terminal signal sequences of 15-30 amino acids. ----These sequences are bound by the signal recognition particle (SRP) ------Proteins then undergo cotranslational export. -SRP does not resume translating protein until encountering FtsY in the membrane

The Ribosome, a Translation Machine

-Ribosomes are composed of two subunits, each of which includes rRNA and proteins. ---In prokaryotes, the subunits are 30S and 50S and combine to form the 70S ribosome. 3XrRNA and proteins The 70S ribosome harbors three binding sites for tRNA: 1. A (acceptor) site: binds incoming aminoacyl-tRNA 2. P (peptidyl-tRNA) site: harbors the tRNA with the growing polypeptide chain 3. E (exit) site: binds a tRNA recently stripped of its polypeptide

tRNA Molecules

-tRNAs are decoder molecules that convert the language of RNA into that of proteins. -tRNAs are shaped like a clover leaf (in 2D) and a boomerang (in 3D). -A tRNA molecule has two functional regions: --Anticodon: hydrogen bonds with the mRNA codon specifying an amino acid ---3′ (acceptor) end: binds the amino acid tRNAs contain a large number of unusual, modified bases.

Initiation of transcription

1. RNA polymerase holoenzyme forms a loosely bound, closed complex with DNA. ((+1) the initial start at the 5 'prime end. ) 2. Closed complex must become an open complex through the unwinding of one helical turn 3. RNA polymerase in the open complex becomes tightly bound to DNA, and so begins transcription ---The first ribonucleoside triphosphate (rNTP) of the new RNA chain is usually a purine (A or G) 4. once the holoenzyme reaches the concesus sequence and starts making mRNA (sigma factor falls off)Sigma dissociates after nine RNA bases have been joined.

Termination of transcription

All bacterial genes use one of two known transcription termination signals: 1. Rho-dependent Relies on a protein called Rho and a strong pause site at the 3′ end of the gene --- rho bind to GC region and contact between Tho and RNA polymerase cause termination. 2. Rho-independent Requires a GC-rich region of RNA, as well as 4-8 consecutive U residues. --- stem loop NursA. the contact between hairpin NusA protein and RNA polymerase causes termination.

Annotating the Genome Sequence

Annotation of the DNA sequence is basically understanding what the sequence means. It requires computers that look for patterns, such as regulatory sequences, open-reading frames (ORFs), and rDNA and tRNA genes. An ORF is a sequence of DNA that encodes an actual polypeptide. --In eukaryotes, finding ORFs is complicated by the presence of introns.

All of the following are true of transcription initiation EXCEPT a) The RNA polymerase holoenzyme binds. b) GTP hydrolysis catalyzes bubble formation. c) The closed complex becomes the open complex. d) The first rNTP is usually a purine. e) Position +1 marks the start of the gene.

B

Chaperones are proteins that a) Export other proteins out of the cell b) Help other proteins fold properly c) Degrade misfolded proteins d) Bind other proteins to inactivate them

B

Many computer programs and resources used to analyze DNA and protein sequences are freely available on the Internet.

BLAST NCBI Multiple Sequence Alignment EBI KEGG Motif Search ExPASy coliBASE Joint Genome Institute

How Do Ribosomes Find theRight Reading Frame?

Every mRNA has three potential reading frames, so how does the ribosome find the right one? ---The upstream, untranslated leader RNA contains a purine-rich sequence with the consensus 5′-AGGAGGU-3′ ----Located 4-8 bases upstream of the start codon in Escherichia coli This Shine-Dalgarno sequence is complementary to a sequence at the 3′ end of 16S rRNA of the 30S subunit (small subunit). (after +1 start site transcription (translation is the ribosome binding site) just because you have two genes in the mRNA does not mean your going to code 2 protein at once- separate it)

Transcription occurs in 3 phases

Initiation: RNA pol holoenzyme binds to the promoter -This is followed by melting of the helix and synthesis of the first nucleotide of the RNA 2. Elongation: the RNA chain is extended 3. Termination: RNA pol detaches from the DNA, after the transcript is made

protein degradation How do cells cope with stress-damaged protein?

Many normal proteins contain degradation signals called degrons. -The N-terminal rule suggests that the N-terminal amino acid of a protein directly correlates with its stability. Proteasomes are protein-degrading machines found in eukaryotes and archaea.-they contain cap, core particle and cap. (heteroheptameric) Bacteria contain ATP-dependent proteases, such as Lon and ClpP. (homoheptameric) -both are ATP dependent -Damaged proteins randomly enter chaperone-based refolding pathways or degradation pathways until the protein is repaired or destroyed. Chaperones attempt to refold misfolded proteins ii. If proteins can't be refolded, they are destroyed by proteases

Protein Export to the Periplasm

Many periplasmic proteins, such as SOD and maltose-binding protein, are delivered to the periplasm by a common pathway called the SecA-dependent general secretion pathway. protein is translater in the cytoplasm and the pre secreated AA is sec B delivers the polypeptyde to sec A, secYEG port (ATP) until the whole polypeptide is pushed out into the periplasm and signal lepB is used. once in the periplasm it needs cheperones to be folded.

The Three Stages of Protein Synthesis

Polypeptide synthesis occurs in three stages: 1. Initiation: brings the two ribosomal subunits together, placing the first amino acid in position 2. Elongation: sequentially adds amino acids as directed by mRNA transcript 3. Termination: releases the completed protein and recycles ribosomal subunits -Each phase requires a number of protein factors and energy in the form of GTP

protein modification and folding

Protein structure may be modified after translation: -N-formyl group may be removed by methionine deformylase. -The entire methionine may be removed by methionyl aminopeptidase. -Acetyl groups or AMP can be attached. -Proteolytic cleavages may activate or inactivate a protein.

bioinformatics

Since 1998, the complete genomes of more than 225 microbial species have been published. This wealth of information has spawned a new discipline called bioinformatics, which is dedicated to comparing genes of different species. Data from bioinformatics enable scientists to make predictions about an organism's physiology and evolutionary development. ---Even without culturing the organism in a lab

The Ribosome Is a "Ribozyme"

The ribosome makes the peptide bonds that stitch amino acids together using a remarkable enzymatic activity called peptidyltransferase. Peptidyltransferase is actually a ribozyme (an RNA molecule that carries out catalytic activity). ---Part of 23S rRNA of the large ribosomal subunit. (Peptide bond is going to create a bond with AA in the A site and Psite. The polypetide side change moves to the A site once the tRNA sites.) While highly conserved, there are differences in rRNA sequences that increase in relation to the evolutionary distance among species. ---So rRNA serves as a molecular clock

example of secreation type I

Type I protein secretion moves certain proteins directly from the cytoplasm to the extracellular environment. An efflux ABC transporter(2XATP binding )

The RNA degradosome is composed of ?

an RNase, an RNA helicase, and two metabolic enzymes ---Recent findings suggest that it is compartmentalizedwithin the cell

mutations in the consensus sequences?

can affect the strength of the promoter Some mutations can cause decreased transcription (called "down mutations"), while others cause increased transcription ("up mutations"). will changes the gene expression

Heat-shock proteins, which help in protein folding, are a type of

chaperonin.

termination of protein synthesis

once the stop codon enter the A site. since nothing happends the RF1 enter the site and peptidyltranferase activates to release the polypeptide. the GTP comes and in and release the rRNA to subunits.