Proteins and Translation Notes

Proteins and Translation

Overview

  • This section reviews protein structure and translation, focusing on what gives proteins their specific shapes and properties.

Learning Objectives

  • Describe the general structures of amino acids, polypeptides, subunits, and proteins.
  • Define protein domain and explain how domains relate to protein folding, functions, and the evolution of genes.
  • Describe the steps in translation elongation and termination and when protein folding begins.
  • Describe how initiation and elongation factors contribute to translation.
  • Compare and contrast eukaryotes and prokaryotes in terms of translation initiation and polysome formation.
  • Explain the directionality of ribosome movement and protein synthesis and how the correct reading frame is determined.
  • Explain how “leaky scanning” for Kozak sequences could impact protein sequences produced from a eukaryotic mRNA.

Protein Review

  • Polypeptides are linear sequences of amino acids linked by peptide bonds.
  • Peptide bonds join the carboxyl end of one amino acid to the amino end of another.
  • Each amino acid has an amino and carboxyl end, and a side chain (R group) unique to that amino acid, giving it specific properties.
  • Proteins are composed of one or more polypeptide subunits.
  • To be functional, a protein must fold into the correct 3-D shape and include the correct cofactors/subunits, as well as any required post-translational modifications.
  • Folding is promoted by non-covalent interactions.
  • Enzymes involved in the folding process often have weak affinity for the ribosome and stay near the exit tunnel (similar to the function of the RNA pol II CTD).

Key components of a polypeptide.

  • Amino terminus (N-terminus)
  • Carboxyl terminus (C-terminus)
  • Side chains
  • Peptide bonds

Examples of amino acids with different side chains:

  • Tyrosine (Tyr)
  • Leucine (Leu)
  • Histidine (His)
  • Aspartic acid (Asp)

Protein Domains

  • Protein domains are the structural units of proteins.
  • A domain usually folds independently and has a particular function.
  • Different proteins can contain the same domain.

DNA Binding Domains

  • Common structural motifs that interact with the major groove of DNA are used by many different DNA-binding proteins.
  • Examples: Zinc finger, Leucine zipper, Helix-loop-helix.
    *How the same DNA-binding domain can be found in multiple different proteins: Different proteins can vary greatly in size and number of polypeptide subunits.
  • One polypeptide could contain more than one domain. One protein often contains many domains with different functions.

Translation of the Genetic Code

  • Translation is a highly conserved and complex process in both eukaryotes and prokaryotes.
  • Divided into three major steps:
    1. Initiation: Ribosome attaches to mRNA and begins translating at the initiation/start codon (AUG \rightarrow methionine)
      • Puts the ribosome in the correct reading frame.
      • Example mRNA sequence: 5’-UUAACC AUG GUU CUC UUU….-3’
    2. Elongation: Polypeptide chain elongation is a series of steps repeated until a stop codon is reached (UAA, UAG, or UGA).
    3. Termination: A stop codon signals the end of elongation and the release of the polypeptide.

Ribosome Structure

  • Each ribosome has three sites for association with tRNAs:
    1. A (aminoacyl) site
    2. P (peptidyl) site
    3. E (exit) site

Peptide Bond Formation During Elongation

  • A new peptide bond forms between the amino group of the incoming amino acid and the C-terminus of the growing chain.

Major Steps in Translation Elongation

  1. AA-tRNA binds to an empty A site.
  2. A new peptide bond is formed between the growing chain and the new amino acid (peptidyl transferase activity).
  3. The large subunit translocates.
  4. The small subunit translocates by 3 nucleotides (codon), resulting in an empty A site that can accept another AA-tRNA.
    • The process repeats until the ribosome reaches a stop codon on mRNA.

Translation Elongation Factors

  • Translation elongation factors are additional proteins that improve the efficiency and accuracy of translation (EF-Tu and EF-G in bacteria).
  • They hydrolyze GTP to drive transitions in the ribosome subunits.

Accuracy Checks:

  • Small subunit rRNA hydrogen bonds with the codon-anticodon.
  • A tight (correct) codon/anticodon pairing triggers a conformational change in the ribosome and hydrolysis of GTP by EF-Tu.
  • EF-Tu then releases the AA-tRNA, freeing it for the addition of the AA to the growing chain.

Translation Initiation

  • Beginning from the AUG start codon allows the ribosome to correctly set the reading frame.
  • AUG codes for methionine, so the initial amino acid at the amino (N) terminus of the polypeptide is always Met (but this can be removed later).
  • The start codon is at the 5’ end of the protein-coding sequence in the RNA.
  • The ribosome moves 5’ to 3’ along the mRNA, and the polypeptide is built N-terminus to C-terminus.
  • In prokaryotes, translation can begin at the 5’ end while RNA polymerase is still synthesizing.
  • Initiation requires proteins called initiator factors: IFs in bacteria and eIFs in eukaryotes.

Translation Initiation in Prokaryotes

  • In prokaryotes, the small ribosomal subunit binds to the first AUG codon guided by a specific sequence of nucleotides upstream: the Shine-Dalgarno Sequence (5’-AGGAGG-3’).
  • The Shine-Dalgarno sequence is complementary to a sequence near the 3’ end of 16S rRNA, positioning the ribosome at the correct spot.
  • Shine-Dalgarno sequences can be located anywhere along the mRNA; therefore, prokaryotic ribosomes can synthesize multiple proteins from a single RNA.

Initiation Factors in Prokaryotes:

  • IF1: Helps with attachment to mRNA.

  • IF2: GTP-binding protein that is required for attachment of the first AA-tRNA.

  • IF3: Prevents premature attachment of the large subunit.

  • In bacteria, the initial AUG methionine is a modified version called N-formylmethionine (fMet).

  • The initiator tRNA (carrying fMet) interacts with AUG at what will be the ‘P’ site of the ribosome.

  • IFs are then released, and the large subunit can bind.

Translation Initiation in Eukaryotes

  • Processed 5’ and 3’ ends are important for translation initiation and nuclear export, helping to ensure only completed mRNA are translated.
  • Eukaryotes have larger ribosomes and require additional proteins (12 eIFs or more), making initiation more complex than in prokaryotes.

eIFs and Their Functions:

  • eIFs bind to the small subunit and are important for:
    • Initiator tRNA (Met) binding the ‘P’ site with GTP-bound eIF2.
    • eIF1s: Inducing a conformational change to allow binding of mRNA.
    • eIF3: Interacting with eIF4G on the mRNA complex.

mRNA eIFs:

  • eIF4E: Binds to the 5’ cap.

  • eIF4A: Has helicase activity that uses ATP hydrolysis to unwind any double-stranded regions in mRNA.

  • eIF4G: Links the 5’ cap and 3’ poly(A) tail, converting mRNA into a circular message and interacting with eIF3 on the small subunit.

  • The small ribosomal subunit (along with eIFs and initiator tRNA) finds the 5’ end of mRNA and scans along until it reaches a Kozak sequence (5’-CCACCAUGC-3’).

  • GTP bound to eIF2 is hydrolyzed and released along with other eIFs.

  • Dissociation of initiation factors allows the large subunit to attach

  • Initiator tRNA anticodon bound to AUG in the ‘P’ site is now ready for elongation.

Kozak Sequence

  • The 5’ to 3’ scanning activity searches for the Kozak sequence (5’-CCACCAUGC-3’).
  • This is a consensus sequence, meaning which nucleotides are found most commonly at each position.
  • The actual sequence can vary slightly, but the more different it is, the less efficient initiation of translation at that AUG will be.

Impact:

  • The difference of the sequence affects the proteins produced from an mRNA.

Translation Termination

  • Translation continues until the ribosome reaches a stop codon for which no corresponding tRNA is available.
  • Release factors (which resemble tRNA and recognize stop codons) bind in the vacant A site and catalyze the addition of water instead of an amino acid, freeing the C-terminus (no longer attached to a tRNA).

Translation - Polyribosomes

  • Multiple translation initiations typically take place on the same mRNA.
  • Multiple ribosomes associated with an mRNA form a polyribosome (or polysome).
  • Attach at AUG, start translating, and once AUG is free, another ribosome can assemble.

Summary of Protein Production in Eukaryotes

  • Previously covered:
    • Maintenance of heritable information (replication/repair of DNA).
    • How the information carried in DNA can be used to produce RNA and proteins that carry out cell functions (transcription and translation).
  • The next focus will be on how this process is regulated to make different cell types/functions.

Review Questions for Practice

Review Questions:

Which of the following are true about the given image?
  • mRNA
  • A) This is more likely to occur in prokaryotes than eukaryotes
  • B) “B” is directly above an RNA polymerase
  • C) Transcription is proceeding in the “A” to “C” direction
  • D) “D” marks the 5’ end of the RNA
  • E) Ribosomes are moving in the ”B” to “E” direction
  • F) Each RNA can only be used to produce 1 copy of the protein it encodes
For each of the following, categorize them as occurring only in prokaryotes, only in eukaryotes, in both, or in neither
  • Translation of an mRNA can begin before transcription of the mRNA is complete
  • The Shine-Dalgarno sequence base pairs with rRNA
  • eIF4E and eIF4G form a complex that creates a circular mRNA structure
  • mRNA is bound by the small ribosomal subunit before the large ribosomal subunit
  • The Kozak sequence is important for initiating translation at a start codon
  • Translation terminates when the stop amino acid is added to the polypeptide chain
  • The initiator tRNA brings in N-formylmethionine as the first amino acid
  • The small ribosomal subunit binds the first tRNA before it binds mRNA
  • The small and large ribosomal subunits do not bind together until they are assembled at a start codon in the mRNA
Place these events in order (and fill in the blanks) for adding an amino acid onto a growing chain during translation in bacteria
  • A new peptide bond is formed
  • The large subunit translocates forward by ____ nucleotides
  • EF-Tu hydrolyzes ____
  • A new charged tRNA base-pairs with the codon in the ____ site
  • The small subunit translocates forward by _nucleotides