Chap 7B - protein synthesis

Up to transcription

Describe termination of transcription in eu

1. mRNA synthesized by transcription alone is pre-mRNA 

2. Pre-mRNA relongates until RNA polymerase reads a terminator sequence on DNA template strand TTATT which codes for a polyadenylation signal (AAUAAA)

3. AAUAAA is transcribed by RNA polymerase 

4. Certain proteins are recruited to cleave the RNA transcript at a point about 10-35 RNA nucleotides downstream from the polyadenylation signal -> halting further elongation

Pre-RNA consists of : Exons and introns + 5’ UTR (UnTranslated region) (a short region before the start codon) + 3’ UTR (short region at the end of the last exon, which includes the polyadenylation signal)

5’ UTR:  facilitates the binding of the small ribosomal subunit on the mRNA -> helps to increase the translational efficiency of the mRNA.

3’ UTR : helps to maintain stability of the mRNA

Describe termination of transcription in pro

• The terminator sequence containing self-complementary DNA sequences is transcribed by RNA polymerase, forming self-complementary sequences in the mRNA

• Complementary base pairing between the self-complementary sequences in mRNA forms a hair-pin loop, causing the dissociation of RNA polymerase and termination of transcription

The resulting mRNA transcript consists of:

1. Protein-coding sequence starting with AUG (*There are no introns in prokaryotes)

2. 5’ UTR (a short region before the start codon that is not translated)

3. 3’ UTR (short region at the end of the last exon that is not translated)

Describe key features of eu protein synthesis (diff from pro protein synthesis) 

1. The presence of introns that are interspersed between exons -> requiring post-transcriptional RNA splicing prior to translation

2. During initiation of transcription, the formation of the transcription initiation complex requires the assembly of proteins known as transcription factors other than RNA polymerase

3. The product of transcription is a pre-mRNA or primary transcript that requires posttranscriptional modifications before translation occurs to produce a polypeptide

3. There is a relatively large number of control elements associated with a eukaryotic gene

Describe PTCM

• pre-mRNA -> mature mRNA 

• BEFORE leaving the nucleus into the cytoplasm to be translated into a polypeptide

• Absent in prokaryotes since they have no introns

• The lack of nuclear envelope in prokaryotes allows the mRNA to be immediately used for translation by ribosomes

Describe capping of PTCM

Terms 

• Exonucleases : enzymes that work by cleaving nucleotides one at a time from end of polynucleotide chain 

Process

• 7-methylguanosine residue , a modified form of guanine is added to 5’ terminal end of pre-mRNA molecule through 5-triphosphate linkage, catalysed by mRNA guanyltranferase 

• This guanine base has an additional methyl group at the 7th position on the guanine ring

Purpose

1. Marks 5’ end of mRNA molecule , signaling to ribosomal subunit to bind for translation

 

2. Prevents degradation of of mRNA by hydrolytic enzymes in the cytoplasm, such as 5’ exonucleases -> more stable template for translation 

• The slower the degradation of the mRNA, the longer it exists to be translated into polypeptide, hence increasing gene expression
  

3. Facilitates the export of mature mRNA

Describe polyadenylation of 3’ end of PTCM

Process	Addition of polyadenylate tail of 50-200 adenine nucleotides to 3’ terminal end of pre-mRNA transcript  Catalyzed by polyadenylate (poly A) polymerase enzyme , which adds multiple adenine nucleotides to 3’ end of pre-mRNA  The poly(A) tail is a binding site for a group of proteins known as the poly(A)-binding proteins
Purpose	Facilitates exports of mRNA into cytoplasm from nucleus via nuclear pores  Prevents degradation of mRNA molecule by both exonucleases and endonucleases -> more stable for translation  More stable the mRNA, the longer it exists in the cytoplasm -> more proteins can be synthesised Helps ribosomes attach to the 5’ end of the mRNA during translation

Describe RNA splicing and alternative RNA splicing of PTCM

Terms	Transcription unit has both introns and exons  Occurs in the nucleus Requires energy from hydrolysis of ATP  Introns sequences : non coding regions (tend to start with ‘GU-’ and end with ‘-AG’)  Exons : coding regions  Presence of exons and introns in genes enables a single gene to encode more than one type of polypeptides Splice sites : located at junction of an intron and exon (GU- and AG-)  Untranslated regions :  5’ UTR : between 5’ modified guanine cap and start codon -> regulate rate of translation 3’ UTR : after stop codon -> regulate rate of translation snRNPs small nuclear ribonucleoproteins = proteins complexed with snRNA) :  located in the nucleus and composed of snRNAs (small nuclear RNAs) and various proteins
Process	snRNPs contain snRNA which bind to splice sites at each end of an intron in pre-mRNA via complementary base pairing -> catalyzes splicing reaction + bringing together splice sites to form a larger complex called spliceosome -> this brings the exons upstream and downstream of an intron close together while the intron is looped into a structure (lariat) -> facilitating the removal of introns (they are excised)  Ligase catalyzes formation of phosphodiester bonds between adjacent exons (they are spliced together) -> creating continuous mature mRNA transcript composed solely of exons
Purpose	Facilitate the transport of mature mRNA from nucleus to the cytoplasm Ensure that only exons are translated to produce functional proteins
Alternative splicing of RNA	Purpose:  Alternative splicing allows the number of different protein products an organism can produce to be greater than its number of genes. As two or more different polypeptide sequences can be derived from one gene, organism can carry fewer genes in its genome -> shorter genome  Process:  1 pre-mRNA spliced into more than 1 type of mature mRNA  Conversion of one type of pre-mRNA into more than one types of mature mRNA by retaining and splicing only certain exons (all introns still excised)  Different combinations of exons are spliced together to give different mature mRNAs

Describe small and large ribosomal subunit of ribosomes

Small ribosomal subunit

Has a mRNA binding site It binds to the mRNA near its 5’ end, via complementary base pairing between the mRNA and rRNA found in the mRNA binding site Prokaryotic mRNA: mRNA binding site contains rRNA that is complementary to the Shine-Dalgarno sequence on the 5’ end of mRNA, allows mRNA binding via complementary base pairing Eukaryotic mRNA: binds to the mRNA binding site on the small subunit via 5’ UTR

Large ribosomal subunit

Contains peptidyl transferase  Has 3 binding sites for tRNA  Exit site : site where empty tRNA without AA occupies prior to exiting ribosome  Peptidyl-tRNA site : occupied by peptidyl-tRNA (tRNA carrying growing polypeptide chain)  Aminoacyl-tRNA site : attachment site for incoming aminoacyl-tRNA